Systems and methods for targeting cancer cells

ABSTRACT

The present disclosure provides an immune cell genetically modified to produce two antigen triggered polypeptides, each recognizing a different cell surface antigen. The present disclosure provides a system two antigen-triggered polypeptides, each recognizing a different cell surface antigen. The present disclosure provides a method of killing a target cancer cell, using a genetically modified immune cell or a system of the present disclosure. The present disclosure provides a computational method to identify target N antigen pairs on a cancer cell.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Patent Application No. 62/333,106, filed May 6, 2016, which application is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. GM071966 and GM071508 awarded by the National Institutes of Health/NIGMS and Grant No. HL117798 awarded by the National Institutes of Health/NHLBI. This invention was also made with government support under Grant No. R01 CA196277 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file, “UCSF-537WO_SeqList_ST25.txt” created on May 5, 2017 and having a size of 37,675 KB. The contents of the text file are incorporated by reference herein in their entirety.

INTRODUCTION

Immune cell activation can be engineered ex vivo through expression of various designer antigen-triggered immune cell receptors including, e.g., synthetic chimeric antigen receptors (CAR), synthetic Notch polypeptides (synNotch), inhibitory CARs (iCARs), split CARs, and engineered T cell Receptors (TCR). A goal of such immune cell activation is targeting and killing of cancer cells in a patient, while avoiding or at least minimizing killing of non-cancerous cells.

SUMMARY

The present disclosure provides an immune cell genetically modified to produce two antigen-triggered polypeptides, each recognizing a different cell surface antigen. The present disclosure provides a system comprising two antigen-triggered polypeptides (or nucleic acids encoding same), each recognizing a different cell surface antigen. The present disclosure provides a method of killing a target cancer cell, using a genetically modified immune cell or a system of the present disclosure. The present disclosure provides a computational method to identify target antigen pairs on a cancer cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides antigen combinations for various cancers.

FIG. 2 provides amino acid sequences of antigens within antigen combinations.

FIG. 3A-3D provide schematic depictions of various antigen-triggered polypeptides; and AND and AND-NOT logic gates using the antigen-triggered polypeptides.

FIG. 4 provides cancer-associated target antigens in clinical trials.

FIG. 5A-5G provide schematic depictions of exemplary synNotch receptor Notch regulatory regions.

FIG. 6A-6F provide schematic depictions of exemplary split CARs.

FIG. 7 provides an example of a decision tree for identifying AND gate antigen pairs.

FIG. 8 provides an example of a decision tree for identifying AND-NOT gate antigen pairs.

FIG. 9 provides clinical antigen AND-gate combinations for various exemplary cancers.

FIG. 10 provides clinical antigen AND NOT-gate combinations for various exemplary cancers.

FIG. 11 provides novel antigen AND-gate combinations for various exemplary cancers.

FIG. 12 provides novel antigen AND NOT-gate combinations for various exemplary cancers.

FIG. 13 provides clinical-clinical antigen combinations for various exemplary cancers.

FIG. 14 provides single antigens useful for various exemplary cancers.

FIG. 15 provides three antigen combinations for various exemplary cancers.

DEFINITIONS

The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

The terms “chimeric antigen receptor” and “CAR”, used interchangeably herein, refer to artificial multi-module molecules capable of triggering or inhibiting the activation of an immune cell which generally but not exclusively comprise an extracellular domain (e.g., a ligand/antigen binding domain), a transmembrane domain and one or more intracellular signaling domains. The term CAR is not limited specifically to CAR molecules but also includes CAR variants. CAR variants include split CARs wherein the extracellular portion (e.g., the ligand binding portion) and the intracellular portion (e.g., the intracellular signaling portion) of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled. CAR variants also include bispecific CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application No. US2014/016527; Fedorov et al. Sci Transl Med (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety.

As used herein, the term “immune cells” generally includes white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow. “Immune cells” includes, e.g., lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells).

“T cell” includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), T-regulatory cells (Treg) and gamma-delta T cells.

A “cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses.

As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc. In some cases, the individual is a human.

A “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a target antigen” includes a plurality of such antigens and reference to “the system” includes reference to one or more systems and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides an immune cell genetically modified to produce two antigen-triggered polypeptides, each recognizing a different cell surface antigen. The present disclosure provides a system of two antigen-triggered polypeptides, each recognizing a different cell surface antigen. The present disclosure provides a method of killing a target cancer cell, using a genetically modified immune cell or a system of the present disclosure.

The present disclosure provides an in vitro genetically modified cytotoxic immune cell, where the cytotoxic immune cell is genetically modified to produce two different antigen-triggered polypeptides that recognize two different cell surface antigens, and where at least one of the two different cell surface antigens is present on the surface of a target cancer cell. In some cases, the two different antigen-triggered polypeptides comprise: a) a first antigen-triggered polypeptide that binds specifically to a first target cell surface antigen present on a target cancer cell; and b) a second antigen-triggered polypeptide that binds specifically to a second target cell surface antigen. In some cases, the genetically modified cytotoxic immune cell is a genetically modified cytotoxic T cell or a genetically modified natural killer cell. In some cases, the two different antigen-triggered polypeptides provide an AND gate; thus, for example, in some cases, the genetically modified cytotoxic immune cell is activated to kill a target cancer cell only when the target cancer cell expresses both of the two different cell surface antigens on its cell surface. In some cases, the two different antigen-triggered polypeptides provide an AND-NOT gate; thus, for example, in some cases, the genetically modified cytotoxic immune cell: a) is activated to kill a target cancer cell that expresses the first target cell surface antigen, but not the second target cell surface antigen, on its cell surface; and b) is inhibited from killing a non-cancerous cell if the non-cancerous cell expresses both the first target cell surface antigen and the second target cell surface antigen on its cell surface.

The present disclosure provides a system for killing a target cancer cell, the system comprising: a) a first antigen-triggered polypeptide that binds specifically to a first target antigen present on the target cancer cell, or a first nucleic acid comprising a nucleotide sequence encoding the first antigen-triggered polypeptide; and b) a second antigen-triggered polypeptide that binds specifically to a second target antigen, or a second nucleic acid comprising a nucleotide sequence encoding the second antigen-triggered polypeptide. In some cases, the system provides an AND gate; thus, for example, in some cases, the first target antigen and the second target antigen are both present on the surface of a target cancer cell. In some cases, the system provides an AND-NOT gate; thus, for example, in some cases: a) the first target antigen and the second target antigen are both present on the surface of a non-cancerous cell; and b) the first target antigen, but not the second target antigen, is present on the surface of a target cancer cell. A system of the present disclosure can be introduced ex vivo into an immune cell obtained from a patient, to generate a modified immune cell; and the modified immune cell can be introduced into the patient from whom the immune cell was obtained.

The present disclosure provides a method of killing a target cancer cell in an individual. In some cases, a method of the present disclosure for killing a target cell in an individual comprises: a) introducing a system of the present disclosure into an immune cell (e.g., a CD8⁺ T cell; an NK cell) obtained from the individual, generating a modified immune cell; and b) administering the modified immune cell to the individual, where the modified immune cell kills the target cancer cell in the individual.

The present disclosure provides a method of killing a target cancer cell in an individual. In some cases, a method of the present disclosure for killing a target cell in an individual comprises administering a genetically modified cytotoxic immune cell (e.g., a genetically modified CD8⁺ T cell; a genetically modified NK cell) of the present disclosure to the individual, where the genetically modified immune cell kills the target cancer cell in the individual.

As noted above, a genetically modified cytotoxic immune cell of the present disclosure, and a system of the present disclosure, involve at least two antigen-triggered polypeptides that recognize two different cell surface antigens. A pair of antigen-triggered polypeptides recognizes and binds to a pair of target antigens; antigen binding activates the antigen-triggered polypeptides. Thus, a first antigen-triggered polypeptide binds a first member of a target antigen pair; and a second antigen-triggered polypeptide binds a second member of the target antigen pair. Target antigen combinations (also referred to herein as “target antigen pairs”) are provided in FIG. 1 and FIG. 9-14 . At least one of the two antigens of a target antigen pair listed in FIG. 1 or FIG. 9-14 is present on the surface of a target cancer cell. In some cases, the second target antigen of a target antigen pair is present on the surface of the same target cancer cell as the first target antigen of the target antigen pair. In some cases, the first target antigen of the target antigen pair is present on the surface of a target cancer cell, and the second target antigen of a target antigen pair is not present on the surface of the same target cancer cell; in these cases, both antigens of the target antigen pair are present on the surface of a non-cancerous cell. The target antigen combinations presented in FIG. 1 and FIG. 9-14 provide for an AND logic gate or an AND-NOT logic gate for a particular cancer cell type.

Where a target antigen pair provides for an AND logic gate, both antigens must be present on the surface of a target cancer cell in order for a genetically modified cytotoxic immune cell of the present disclosure to kill the target cancer cell, where in this case the genetically modified cytotoxic immune cell is genetically modified to express two antigen-triggered polypeptides, each recognizing one of the target antigens of the target antigen pair. For example, where a target antigen pair present in FIG. 1 and/or FIG. 9-14 is indicated as providing an AND logic gate, both target antigens of the target antigen pair must be present on the surface of a target cancer cell in order for a genetically modified cytotoxic immune cell of the present disclosure to kill the target cancer cell; and the genetically modified cytotoxic immune cell must express both a first antigen-triggered polypeptide that specifically binds the first target antigen of the target antigen pair and a second triggered polypeptide that specifically binds the second antigen of the target antigen pair. For example, in some cases, expression of the second antigen-triggered polypeptide is induced when the first antigen-triggered polypeptide binds to the first target antigen of the target antigen pair.

Where a target antigen pair provides an AND-NOT logic gate, a genetically modified cytotoxic immune cell of the present disclosure: a) is activated to kill a target cancer cell that expresses the first target cell surface antigen, but not the second target cell surface antigen, on its cell surface; and b) is inhibited from killing a non-cancerous cell if the non-cancerous cell expresses both the first target cell surface antigen and the second target cell surface antigen on its cell surface; in these cases, the genetically modified cytotoxic immune cell must express both a first antigen-triggered polypeptide that specifically binds the first target antigen of the target antigen pair and a second triggered polypeptide that specifically binds the second antigen of the target antigen pair. For example, in some cases, binding of the second antigen-triggered polypeptide to the second target cell surface antigen (expressed on a non-cancerous cell) inhibits T cell activation that would normally be induced by binding of the first antigen-triggered polypeptide to the first target antigen (present on the cancer cell surface and on the non-cancerous cell surface). In this manner, unintended/undesired killing of a non-cancerous cell is reduced, because the target cancer cell expressing the first target antigen and not the second target antigen will be preferentially killed over the non-cancerous cell expressing both the first target antigen and the second target antigen. Since the cancer cell does not express the second target cell surface antigen (expressed on a non-cancerous cell), binding of the first antigen-triggered polypeptide to the first target antigen (present on the cancer cell surface) results in activation of the genetically modified cytotoxic T cell and killing of the cancer cell.

In some cases, the first antigen-triggered polypeptide is a synNotch receptor and the second antigen-triggered polypeptide is a chimeric antigen receptor (CAR). In some cases, the first antigen-triggered polypeptide is a synNotch receptor and the second antigen-triggered polypeptide is a T cell receptor (TCR). In some cases, the first antigen-triggered polypeptide is a synNotch receptor, and the second antigen-triggered polypeptide is a split CAR (e.g., an ON-switch CAR). In some cases, the first antigen-triggered polypeptide is a synNotch receptor, and the second antigen-triggered polypeptide is one polypeptide chain of a split CAR (e.g., an ON-switch CAR). In some cases, the first antigen-triggered polypeptide is a synNotch receptor, and the second antigen-triggered polypeptide is another synNotch receptor. Any or either of the first and second antigen-triggered polypeptides of the subject systems may independently be a synNotch receptor, a CAR, a TCR or the like. In some cases, both the first and second antigen-triggered polypeptides of the subject systems may be a synNotch receptor, a CAR, a TCR or the like.

In some cases, the first antigen-triggered polypeptide is a CAR, and the second antigen-triggered polypeptide is an antigen-binding inhibitory polypeptide, such as e.g. an inhibitory CAR (iCAR). In some cases, the first antigen-triggered polypeptide is a TCR, and the second antigen-triggered polypeptide is an antigen-binding inhibitory polypeptide, such as e.g. an iCAR. In some cases, the first antigen-triggered polypeptide is an ON-switch CAR, and the second antigen-triggered polypeptide is an antigen-binding inhibitory polypeptide, such as e.g. an iCAR. In some cases, the first antigen-triggered polypeptide is a CAR, and the second antigen-triggered polypeptide is a synNotch receptor. In some cases, the first antigen-triggered polypeptide is a TCR, and the second antigen-triggered polypeptide is a synNotch receptor. In some cases, the first antigen-triggered polypeptide is an ON-switch CAR, and the second antigen-triggered polypeptide is a synNotch receptor.

In some cases, the target cancer cell is a liposarcoma, a glioblastoma, a breast cancer cell, a renal cancer cell, a pancreatic cancer cell, a melanoma, an anaplastic lymphoma, a leiomyosarcoma, an astrocytoma, an ovarian cancer cell, a neuroblastoma, a mantle cell lymphoma, a sarcoma, a non-small cell lung cancer cell, an AML cell, a stomach cancer cell, a B-cell cancer cell, a lung cancer cell, or an oligodendroglioma.

AND Gate Target Antigen Pairs

As noted above, in some cases, expression of the second antigen-triggered polypeptide in a genetically modified immune cell is induced only when the first antigen-triggered polypeptide binds to the first target antigen of the target antigen pair, where the binding to the first target antigen activates the first antigen-triggered polypeptide. Non-limiting examples of 2-input AND gates (AND gates based on 2 target antigens) and 3-input AND gates (AND gates based on 3 target antigens) are depicted schematically in FIG. 3A-3B.

For example, in some cases, the first antigen-triggered polypeptide is a synNotch receptor and activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces expression of the second antigen-triggered polypeptide. The second antigen-triggered polypeptide binds to the second antigen of the target antigen pair, where the second antigen is expressed on the surface of the target cancer cell. As an example, in some cases, the first antigen-triggered polypeptide is a synNotch receptor and the second antigen-triggered polypeptide is a single chain CAR. As another example, in some cases, the first antigen-triggered polypeptide is a synNotch receptor and the second antigen-triggered polypeptide is a TCR. For example, in some cases, the synNotch polypeptide comprises an intracellular domain comprising a transcriptional activator, and activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces release of the transcriptional activator; the released transcriptional activator activates transcription of the TCR or the single-chain CAR.

As another example, in some cases, the first antigen-triggered polypeptide is a synNotch receptor and activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces expression of the second antigen-triggered polypeptide, where the second antigen-triggered polypeptide is a heterodimeric (“two chain” or “split”) CAR comprising a first polypeptide chain and a second polypeptide chain. The heterodimeric CAR binds to the second antigen of the target antigen pair, where the second antigen is expressed on the surface of the target cancer cell. For example, in some cases, the first antigen-triggered polypeptide is a synNotch receptor and the second antigen-triggered polypeptide is a split CAR (e.g., an ON-switch CAR). In some cases, activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces expression of only the first polypeptide chain of the heterodimeric CAR; expression of the second polypeptide chain of the heterodimeric CAR can be constitutive. For example, in some cases, the synNotch polypeptide comprises an intracellular domain comprising a transcriptional activator, and activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces release of the transcriptional activator; the released transcriptional activator activates transcription of the first polypeptide chain of the heterodimeric CAR. In some cases, activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces expression of only the second polypeptide chain of the heterodimeric CAR; expression of the first polypeptide chain of the heterodimeric CAR can be constitutive. Once the first polypeptide chain of the heterodimeric CAR is produced in the cell, it heterodimerizes with the second polypeptide chain of the heterodimeric CAR. As another example, in some cases, the synNotch polypeptide comprises an intracellular domain comprising a transcriptional activator, and activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces release of the transcriptional activator; the released transcriptional activator activates transcription of the second polypeptide chain of the heterodimeric CAR.

In AND gate systems, unintended/undesired killing of non-target cells is reduced; for example, a cell that expresses on its cell surface only one of the target antigen pair is not killed by a genetically modified cytotoxic immune cell of the present disclosure.

In some cases, a genetically modified immune cell or a system of the present disclosure provides for a 3-input AND gate, comprising: 1) a first target antigen of an AND-gate target antigen pair depicted in FIG. 1 or FIG. 9-14 ; 2) a second target antigen of the AND-gate target antigen pair; and 3) a third target antigen, where the third target antigen is a target antigen depicted in FIG. 4 or Table 3, where e.g., the third target antigen may be a target antigen for the same cancer cell type as the target antigen pair. As one non-limiting example, where the target AND-gate antigen pair is SYT11 AND TNFRSF17 (for a glioma cell) (as shown in FIG. 1 ), a suitable third target antigen is EphA2 (as shown in FIG. 4 ).

AND-NOT Gate Target Antigen Pairs

As noted above, in some cases, where a target antigen pair provides an AND-NOT logic gate, a genetically modified cytotoxic immune cell of the present disclosure: a) is activated to kill a target cancer cell that expresses the first target cell surface antigen, but not the second target cell surface antigen, on its cell surface; and b) is inhibited from killing a non-cancerous cell if the non-cancerous cell expresses both the first target cell surface antigen and the second target cell surface antigen on its cell surface; in these cases, the genetically modified cytotoxic immune cell must express both a first antigen-triggered polypeptide that specifically binds the first target antigen of the target antigen pair and a second triggered polypeptide that specifically binds the second antigen of the target antigen pair. Non-limiting examples of 2-input AND-NOT gates (AND-NOT gates based on 2 target antigens) and 3-input AND-NOT gates (AND-NOT gates based on 3 target antigens) are depicted schematically in FIG. 3C-3D.

As an example, in some cases, the first antigen-triggered polypeptide is a CAR, and the second antigen-triggered polypeptide is an iCAR. Binding of the iCAR to the second antigen (present on the surface of a non-cancerous cell, but not on the surface of a target cancer cell) of a target antigen pair inhibits T-cell activation mediated by activation of the CAR upon binding to the first antigen (present on the surface of the target cancer cell and on the surface of the non-cancerous cell) of the target antigen pair. As another example, in some cases, the first antigen-triggered polypeptide is a TCR, and the second antigen-triggered polypeptide is an iCAR. Binding of the iCAR to the second antigen (present on the surface of a non-cancerous cell, but not on the surface of a target cancer cell) of a target antigen pair blocks or reduces T-cell activation mediated by activation of the TCR upon binding to the first antigen (present on the surface of the target cancer cell and on the surface of the non-cancerous cell) of the target antigen pair.

The above provides examples of AND-NOT gates where the inhibitory component is an iCAR; however, as will be readily understood, the inhibitory components of combinatorial antigen gates having “NOT” functionality are not so limited and may generally include any polypeptide configured to inhibit an activity, e.g., an activity induced by binding of a first activating antigen in an AND-NOT gate, including where such inhibition is conferred through the presence of an inhibitory domain. Inhibitory components of combinatorial antigen gates having “NOT” functionality may be specific for an antigen present on a non-target cell, including e.g., where such antigen is absent or present in low amounts on the surface of a target cell.

As another example, in some cases, the first antigen-triggered polypeptide is a CAR, and the second antigen-triggered polypeptide is a synNotch polypeptide comprising an intracellular domain that, when released upon activation of the synNotch polypeptide by binding to the second target antigen, induces expression of an intracellular inhibitor that inhibits T-cell activation mediated by activation of the CAR upon binding to the first antigen (present on the surface of the target cancer cell and on the surface of the non-cancerous cell) of the target antigen pair. As another example, in some cases, the first antigen-triggered polypeptide is a TCR, and the second antigen-triggered polypeptide is a synNotch polypeptide comprising an intracellular domain that, when released upon activation of the synNotch polypeptide by binding to the second target antigen, induces expression of an intracellular inhibitor that inhibits T-cell activation mediated by activation of the TCR upon binding to the first antigen (present on the surface of the target cancer cell and on the surface of the non-cancerous cell) of the target antigen pair.

As another example, in some cases, the first antigen-triggered polypeptide is a CAR, and the second antigen-triggered polypeptide is a synNotch polypeptide comprising an intracellular domain that, when released upon activation of the synNotch polypeptide by binding to the second target antigen, induces expression of an extracellular inhibitor that inhibits T-cell activation mediated by activation of the CAR upon binding to the first antigen (present on the surface of the target cancer cell and on the surface of the non-cancerous cell) of the target antigen pair. As another example, in some cases, the first antigen-triggered polypeptide is a TCR, and the second antigen-triggered polypeptide is a synNotch polypeptide comprising an intracellular domain that, when released upon activation of the synNotch polypeptide by binding to the second target antigen, induces expression of an extracellular inhibitor that inhibits T-cell activation mediated by activation of the TCR upon binding to the first antigen (present on the surface of the target cancer cell and on the surface of the non-cancerous cell) of the target antigen pair.

In some cases, a genetically modified immune cell or a system of the present disclosure provides for a 3-input AND, AND-NOT gate, comprising: 1) a first target antigen of an AND-NOT gate target antigen pair depicted in FIG. 1 or FIG. 9-14 ; 2) a second target antigen of the AND-NOT gate target antigen pair; and 3) a third target antigen, where the third target antigen is a target antigen depicted in FIG. 4 or Table 3, including e.g., where the third target antigen may be a target antigen for the same cancer cell type as the target antigen pair. In some cases, the 3-input AND, AND-NOT gate comprises: 1) the first target antigen of an AND-NOT gate target antigen pair depicted in FIG. 1 or FIG. 9-14 ; AND-NOT; 2) the second target antigen of the AND-NOT gate target antigen pair; AND 3) the third target antigen. In some cases, the 3-input AND, AND-NOT gate comprises: 1) the second target antigen of an AND-NOT gate target antigen pair depicted in FIG. 1 or FIG. 9-14 ; AND-NOT 2) the first target antigen of the AND-NOT gate target antigen pair; AND 3) the third target antigen.

As one non-limiting example, where the target AND-NOT gate antigen pair is NLGN1 AND-NOT TNFRSF17 (for a glioma cell) (as shown in FIG. 1 ), a suitable third target antigen is EphA2 (as shown in FIG. 4 ).

Antigen-Triggered Polypeptides

As noted above, an antigen-triggered polypeptide can be a synNotch polypeptide; a CAR; or a TCR. A CAR can be an ON-switch (“split”) CAR, a single-chain CAR, an iCAR, etc. Schematic depictions of examples of antigen-triggered polypeptides are provided in FIG. 5 and FIG. 6A-6F.

synNotch Polypeptides

As noted above, in some cases an antigen-triggered polypeptide produced in a genetically modified immune cell of the present disclosure, or present in a system of the present disclosure, or encoded by a nucleotide sequence in a nucleic acid present in a system of the present disclosure, is a synNotch receptor (a “synNotch polypeptide”). synNotch polypeptides are described in PCT/US16/19188, the disclosure of which is incorporated herein by reference in its entirety. Schematic depictions of exemplary synNotch polypeptides are provided in FIG. 5A-5G.

In some cases, a synNotch polypeptide is a chimeric Notch polypeptide comprising, from N-terminus to C-terminus and in covalent linkage: a) an extracellular domain comprising an antigen binding member that is not naturally present in a Notch receptor polypeptide and that specifically binds to an antigen; b) a Notch regulatory region comprising a Lin 12-Notch repeat, an S2 proteolytic cleavage site, and a transmembrane domain comprising an S3 proteolytic cleavage site; c) an intracellular domain comprising a transcriptional activator or a transcriptional repressor that is heterologous to the Notch regulatory region and replaces a naturally-occurring intracellular Notch domain, wherein binding of the antigen binding member to the antigen, present on a cell or other solid support, induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain.

In some cases, a synNotch polypeptide is a chimeric Notch polypeptide comprising, from N-terminus to C-terminus and in covalent linkage: a) an extracellular domain comprising a single-chain Fv (scFv) or a nanobody that specifically binds to an antigen; b) a Notch regulatory region comprising a Lin 12-Notch repeat, a heterodimerization domain comprising an S2 proteolytic cleavage site and a transmembrane domain comprising an S3 proteolytic cleavage site; and c) an intracellular domain, heterologous to the Notch regulatory region, comprising a transcriptional activator comprising a DNA binding domain, wherein the transcriptional activator replaces a naturally-occurring intracellular notch domain, and wherein binding of the scFv or the nanobody to the antigen in trans induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain and wherein the chimeric Notch polypeptide does not bind its naturally-occurring ligand Delta.

In some cases, a synNotch polypeptide comprises, from N-terminal to C-terminal and in covalent linkage: a) an extracellular domain that specifically binds to an antigen (e.g., a single-chain Fv (scFv), a nanobody, and the like); b) a Notch receptor polypeptide comprising one or more proteolytic cleavage sites and having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a Notch receptor polypeptide as described below; and c) an intracellular domain comprising a transcriptional regulator (e.g., a transcriptional activator or a transcriptional repressor). When the synNotch polypeptide is present in a cell, binding of the extracellular domain (e.g., the scFv or the nanobody) to the antigen induces cleavage of the Notch receptor polypeptide at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain. In some cases, the intracellular domain comprises a transcriptional activator. In some cases, the intracellular domain comprises a transcriptional repressor.

In some cases, a Notch receptor polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of a Notch receptor including e.g., any of SEQ ID NOs: 7240-7245 and 7468-7475. In some instances, the Notch regulatory region of a Notch receptor polypeptide is a mammalian Notch regulatory region, including but not limited to e.g., a mouse Notch (e.g., mouse Notch1, mouse Notch2, mouse Notch3 or mouse Notch4) regulatory region, a rat Notch regulatory region (e.g., rat Notch1, rat Notch2 or rat Notch3), a human Notch regulatory region (e.g., human Notch1, human Notch2, human Notch3 or human Notch4), and the like or a Notch regulatory region derived from a mammalian Notch regulatory region and having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of a mammalian Notch regulatory region of a mammalian Notch receptor amino acid sequence, including e.g., SEQ ID NOs: 7240-7245 and 7468-7475.

Subject Notch regulatory regions may include or exclude various components (e.g., domains, cleavage sites, etc.) thereof. Examples of such components of Notch regulatory regions that may be present or absent in whole or in part, as appropriate, include e.g., one or more EGF-like repeat domains, one or more Lin12/Notch repeat domains, one or more heterodimerization domains (e.g., HD-N or HD-C), a transmembrane domain, one or more proteolytic cleavage sites (e.g., a furin-like protease site (e.g., an S1 site), an ADAM-family protease site (e.g., an S2 site) and/or a gamma-secretase protease site (e.g., an S3 site)), and the like. Notch receptor polypeptides may, in some instances, exclude all or a portion of one or more Notch extracellular domains, including e.g., Notch-ligand binding domains such as Delta-binding domains. Notch receptor polypeptides may, in some instances, include one or more non-functional versions of one or more Notch extracellular domains, including e.g., Notch-ligand binding domains such as Delta-binding domains. Notch receptor polypeptides may, in some instances, exclude all or a portion of one or more Notch intracellular domains, including e.g., Notch Rbp-associated molecule domains (i.e., RAM domains), Notch Ankyrin repeat domains, Notch transactivation domains, Notch PEST domains, and the like. Notch receptor polypeptides may, in some instances, include one or more non-functional versions of one or more Notch intracellular domains, including e.g., non-functional Notch Rbp-associated molecule domains (i.e., RAM domains), non-functional Notch Ankyrin repeat domains, non-functional Notch transactivation domains, non-functional Notch PEST domains, and the like.

In some cases, a Notch receptor polypeptide has a length of from about 310 amino acids (aa) to about 320 aa (e.g., 310 aa, 311 aa, 312 aa, 313 aa, 314 aa, 315 aa, 316 aa, 317 aa, 318 aa, 319 aa, or 320 aa).

In some cases, a Notch receptor polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

(SEQ ID NO: 7246) PPQIEEACELPECQVDAGNKVCNLQCNNHACGW DGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDS QCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHF SDGHCDQGCNSAECEWDGLDCAEHVPERLAAGT LVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVF KRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWA TSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDN RQCVQSSSQCFQSATDVAAFLGALASLGSLNIP YKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLF FVGCGVLLS; NO:7246); and has a length of from 300 amino acids to 310 amino acids (e.g., 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, or 310 amino acids).

In some cases, a Notch receptor polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILDYSFTGGAGRDIPPPQIEEACELPECQ VDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLF DGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVV LLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSS LLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKI EAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLS (SEQ ID NO:7247); and has a length of from 350 amino acids to 370 amino acids (e.g., 350 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, or 370 amino acids).

In some cases, the intracellular domain comprises a transcriptional activator, and release of the intracellular domain causes the transcriptional activator to induce expression of an endogenous gene product in a cell. In some cases, the intracellular domain comprises a transcriptional activator, and release of the intracellular domain causes the transcriptional activator to induce expression of a heterologous gene product (e.g., a CAR; a TCR; a therapeutic antibody). For example, in some cases, a transcriptional control element, responsive to the transcriptional activator, is operably linked to a nucleotide sequence encoding a CAR or a TCR. As another example, a transcriptional control element, responsive to the transcriptional activator, is operably linked to a nucleotide sequence encoding a therapeutic antibody for the treatment of cancer.

In some cases, the intracellular domain comprises a transcriptional activator, and release of the intracellular domain causes the transcriptional activator to induce expression of an intracellular inhibitor that inhibits T-cell activation. In some cases, the intracellular domain comprises a transcriptional activator, and release of the intracellular domain causes the transcriptional activator to induce expression of an inhibitor that is secreted from the cell (an extracellular inhibitor), where the extracellular inhibitor inhibits T-cell activation.

In some cases, the Notch receptor polypeptide comprises, at its N-terminus, one or more epidermal growth factor (EGF) repeats. In some cases, the Notch receptor polypeptide comprises, at its N-terminus, 2 to 11 EGF repeats; e.g., in some cases, the Notch receptor polypeptide comprises, at its N-terminus, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 epidermal growth factor (EGF) repeats. An EGF repeat can have a length of from 35 amino acids (aa) to 45 aa (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 aa). An EGF repeat can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following sequence: PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCH (SEQ ID NO:7248); and can have a length of from 35 amino acids to 40 amino acids (e.g., 35, 36, 37, 38, 39, or 40 amino acids. An EGF repeat can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following sequence: PCLGGNPCYNQGTCEPTSESPFYRCLCPAKFNGLLCH (SEQ ID NO:7249); and can have a length of from 37 amino acids to 40 amino acids (e.g., 37, 38, 39, or 40 amino acids.

In some cases, the Notch receptor polypeptide comprises a synthetic linker between the one or more EGF repeats and the one or more proteolytic cleavage sites.

In some cases, the Notch receptor polypeptide has a length from 50 amino acids to 1000 amino acids. For example, in some cases, the Notch receptor polypeptide has a length of from 50 amino acids (aa) to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to 700 aa, from 700 aa to 800 aa, from 800 aa to 900 aa, or from 900 aa to 1000 aa. In some cases, the Notch receptor polypeptide has a length from 300 amino acids to 400 amino acids.

In some cases, the one or more proteolytic cleavage sites comprises an S2 proteolytic cleavage site, an S3 proteolytic cleavage site or a combination thereof. In some cases, an S3 proteolytic cleavage site comprises the amino acid sequence VLLS (SEQ ID NO:7250). In some cases, an S3 proteolytic cleavage site comprises the amino acid sequence GVLLS (SEQ ID NO:7251). In some cases, the one or more proteolytic cleavage sites comprises an S2 proteolytic cleavage site that is an ADAM family type protease cleavage site, such as e.g., an ADAM-17-type protease cleavage site comprising an Ala-Val dipeptide sequence. In some cases, the one or more proteolytic cleavage sites comprises an S3 proteolytic cleavage site that is a gamma-secretase (γ-secretase) cleavage site comprising a Gly-Val dipeptide sequence. In some cases, the one or more proteolytic cleavage sites further comprises an S1 proteolytic cleavage site. In some cases, the S1 proteolytic cleavage site is a furin-like protease cleavage site comprising the amino acid sequence Arg-X-(Arg/Lys)-Arg, where X is any amino acid. In some cases, the Notch receptor polypeptide does not include an S1 proteolytic cleavage site.

CARs

As noted above, in some cases an antigen-triggered polypeptide produced in a genetically modified immune cell of the present disclosure, or present in a system of the present disclosure, or encoded by a nucleotide sequence in a nucleic acid present in a system of the present disclosure, is a chimeric antigen receptor. Schematic depictions of split CARs are provided in FIG. 6A-6F.

The terms “chimeric antigen receptor” and “CAR”, used interchangeably herein, refer to artificial multi-module molecules capable of triggering or inhibiting the activation of an immune cell which generally but not exclusively comprise an extracellular domain (e.g., a ligand/antigen binding domain), a transmembrane domain and one or more intracellular signaling domains. The term CAR is not limited specifically to CAR molecules but also includes CAR variants. CAR variants include split CARs wherein the extracellular portion (e.g., the ligand binding portion) and the intracellular portion (e.g., the intracellular signaling portion) of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled. CAR variants also include bispecific CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application No. US2014/016527; Fedorov et al. Sci Transl Med (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety.

Spit CAR may be extracellularly split or intracellularly split and may or may not be conditionally heterodimerizable. For example, split CAR systems that are not conditionally heterodimerizable may contain a constitutive heterodimerization domain or other binding pair (e.g., a Fc binding pair or other orthogonal binding pair) that does not depend on the presence of one or more additional molecules for the heterodimerization that results in the formation of an active CAR from assembly of the split portions.

In some instances, an extracellularly split CAR may be split extracellularly at the antigen binding domain into two parts including e.g., where the first part of the split CAR contains an extracellular Fc binding domain that specifically binds to second part of the split CAR that contains the antigen recognition domain.

In some instances, an extracellularly split CAR may be split extracellularly at the antigen binding domain into two parts including e.g., where the first part of the split CAR contains an first part of an orthogonal protein binding pair that specifically binds to the second part of the orthogonal protein binding pair that is contained in the second part of the split CAR that contains the antigen recognition domain.

In some instances, an intracellularly split CAR may be split intracellularly proximal to the transmembrane domain into two parts including e.g., where the first part of the split CAR includes the antigen recognition domain, a transmembrane domain and an intracellular first portion of a constitutive heterodimerization domain and the second part of the split CAR includes a transmembrane domain, the second portion of the constitutive heterodimerization domain proximal to the transmembrane domain, one or more co-stimulatory domains and one or more signaling domains (e.g., ITAM domains).

In some instances, an intracellularly split CAR may be split into two parts intracellularly proximal to an intracellular domain or between two intracellular domains including e.g., where the first part of the split CAR includes the antigen recognition domain, a transmembrane domain, one or more co-stimulatory domains and an intracellular first portion of a constitutive heterodimerization domain and the second part of the split CAR includes a transmembrane domain, one or more co-stimulatory domains, one or more signaling domains (e.g., ITAM domains) and the second portion of the constitutive heterodimerization domain between the one or more co-stimulatory domains and the one or more signaling domains.

In some instances, an intracellularly split CAR may be split into two parts intracellularly between intracellular domains including e.g., where the first part of the split CAR includes the antigen recognition domain, a transmembrane domain, one or more co-stimulatory domains and an intracellular first portion of a constitutive heterodimerization domain proximal to the intracellular terminus of the first part of the split CAR and the second part of the split CAR includes a transmembrane domain, one or more signaling domains (e.g., ITAM domains) and the second portion of the constitutive heterodimerization domain between the transmembrane domain and the one or more signaling domains.

An inhibitory CAR (iCAR) expressed on an immunoresponsive cell specifically binds to an antigen, whereupon binding its antigen the iCAR inhibits the immunoresponsive cell. By “inhibits an immunoresponsive cell” or “suppresses an immunoresponsive cell” is meant induction of signal transduction or changes in protein expression in the cell resulting in suppression of an immune response (e.g., decrease in cytokine production).

Generally, but not exclusively, an iCAR is employed as a component of a bispecific CAR system where the activity of an immunostimulatory CAR (e.g., a CAR or CAR variant) is repressed by the iCAR upon binding of the iCAR to its antigen. An iCAR will generally include an extracellular domain that binds an antigen; a transmembrane domain operably linked to the extracellular domain; and an intracellular domain that activates intracellular signaling to decrease an immune response, the intracellular domain operably linked to the transmembrane domain. In some embodiments, the intracellular signaling domain is selected from the group consisting of a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, and a BTLA polypeptide. In certain embodiments, the transmembrane domain is selected from the group consisting of a CD4 polypeptide, a CD8 polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, and a BTLA polypeptide. In some instances, an iCAR, as described herein, may be or may be derived from one or more of the iCARs described in U.S. Patent Application Publication No. 20150376296, the disclosure of which is incorporated herein by reference in its entirety.

Any convenient extracellular binding domain (i.e., antigen binding domain) may find use in an iCAR including but not limited to e.g., a Fab, scFv, a monovalent or polyvalent ligand, etc., provided the domain is sufficient for specific binding of the iCAR to its antigen. In the contexts of therapy, the antigen binding domain of an iCAR will generally bind a healthy cell antigen in order to repress an immune response that may be otherwise triggered by presentation of a target antigen on the surface of a healthy cell. For example, in the contexts of cancer therapy, the antigen binding domain of an iCAR will generally bind a non-tumor or healthy cell antigen. For example, the extracellular domain may be a binding domain that does not bind one or more tumor antigens, including but not limited to e.g., CD19, CAIX, CEA, CD5, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, a cytomegalovirus (CMV) infected cell antigen, EGP-2, EGP-40, EpCAM, erb-B2,3,4, F8P, Fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-.alpha.2, K-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, Mesothelin, Muc-1, Muc-16, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, and the like. Antigens to which the extracellular domain of an iCAR does not bind are, however, not limited to cancer antigens and may likewise exclude any target antigen to which an immunostimulatory CAR is directed.

In some instances, the antigen binding domain of an iCAR will bind a non-tumor antigen including but not limited to e.g., CD33, CD38, a human leukocyte antigen (HLA), an organ specific antigen, a blood-brain barrier specific antigen, an Epithelial-mesenchymal transition (EMT) antigen, E-cadherin, cytokeratin, Opioid-binding protein/cell adhesion molecule (OPCML), HYLA2, Deleted in Colorectal Carcinoma (DCC), Scaffold/Matrix attachment region-binding protein 1 (SMAR1), cell surface carbohydrate, mucin type O-glycan, etc.

In certain instances, an antigen to which the extracellular domain of an iCAR binds may be an antigen listed in Table 2, provided as FIG. 2 . In some cases, an antigen to which the extracellular domain of an iCAR binds may be an antigen listed in Table 1, provided as FIG. 1 , or in FIG. 9-14 , which antigen is described as the “NOT” portion of an antigen logic gate.

TCRs

As noted above, in some cases an antigen-triggered polypeptide produced in a genetically modified immune cell of the present disclosure, or present in a system of the present disclosure, or encoded by a nucleotide sequence in a nucleic acid present in a system of the present disclosure, is a T-cell receptor (TCR).

A TCR generally includes an alpha chain and a beta chain; and recognizes antigen when presented by a major histocompatibility complex. In some cases, the TCR is an engineered TCR. Any engineered TCR having immune cell activation function can be induced using a method of the present disclosure. Such TCRs include, e.g., antigen-specific TCRs, Monoclonal TCRs (MTCRs), Single chain MTCRs, High Affinity CDR2 Mutant TCRs, CD1-binding MTCRs, High Affinity NY-ESO TCRs, VYG HLA-A24 Telomerase TCRs, including e.g., those described in PCT Pub Nos. WO 2003/020763, WO 2004/033685, WO 2004/044004, WO 2005/114215, WO 2006/000830, WO 2008/038002, WO 2008/039818, WO 2004/074322, WO 2005/113595, WO 2006/125962; Strommes et al. Immunol Rev. 2014; 257(1):145-64; Schmitt et al. Blood. 2013; 122(3):348-56; Chapuls et al. Sci Transl Med. 2013; 5(174):174ra27; Thaxton et al. Hum Vaccin Immunother. 2014; 10(11):3313-21 (PMID:25483644); Gschweng et al. Immunol Rev. 2014; 257(1):237-49 (PMID:24329801); Hinrichs et al. Immunol Rev. 2014; 257(1):56-71 (PMID:24329789); Zoete et al. Front Immunol. 2013; 4:268 (PMID:24062738); Marr et al. Clin Exp Immunol. 2012; 167(2):216-25 (PMID:22235997); Zhang et al. Adv Drug Deliv Rev. 2012; 64(8):756-62 (PMID:22178904); Chhabra et al. Scientific World Journal. 2011; 11:121-9 (PMID:21218269); Boulter et al. Clin Exp Immunol. 2005; 142(3):454-60 (PMID:16297157); Sami et al. Protein Eng Des Sel. 2007; 20(8):397-403; Boulter et al. Protein Eng. 2003; 16(9):707-11; Ashfield et al. IDrugs. 2006; 9(8):554-9; Li et al. Nat Biotechnol. 2005; 23(3):349-54; Dunn et al. Protein Sci. 2006; 15(4):710-21; Liddy et al. Mol Biotechnol. 2010; 45(2); Liddy et al. Nat Med. 2012; 18(6):980-7; Oates, et al. Oncoimmunology. 2013; 2(2):e22891; McCormack, et al. Cancer Immunol Immunother. 2013 April; 62(4):773-85; Bossi et al. Cancer Immunol Immunother. 2014; 63(5):437-48 and Oates, et al. Mol Immunol. 2015 October; 67(2 Pt A):67-74; the disclosures of which are incorporated herein by reference in their entirety.

Antigen-Binding Inhibitory Polypeptides

In some cases an antigen-triggered polypeptide produced in a genetically modified immune cell of the present disclosure, or present in a system of the present disclosure, or encoded by a nucleotide sequence in a nucleic acid present in a system of the present disclosure, may be an inhibitory polypeptide. The term “antigen-binding inhibitory polypeptide”, as used herein, will generally describe a polypeptide, specific for an antigen, that upon binding the antigen inhibits the activity of a second polypeptide (e.g., an activating antigen-specific polypeptide, such as a CAR or TCR or other synthetic stimulatory immune cell receptor) and/or an activity of a cell (e.g., immune activation). iCARs, as described above, are an example of an antigen-binding inhibitory polypeptide; however, the term antigen-binding inhibitory polypeptide is not so limited.

Antigen-binding inhibitory polypeptides will vary and will generally function to mediate repression of an activated or activatable immune cell, including e.g., an immune cell expressing a stimulatory receptor, such as a CAR or TCR. An antigen-binding inhibitory polypeptide will include an inhibitory domain that functions to repress immune cell activation, including e.g., immune cell activation attributed to a stimulatory receptor, such as a CAR or TCR. Domains useful as inhibitory domains will vary depending on the particular context of immune cell activation and repression, including e.g., the particular type of activated cell to be repressed and the desired degree of repression. Exemplary non-limited examples of inhibitory domains include but are not limited to domains and motifs thereof derived from immune receptors including, e.g., co-inhibitory molecules, immune checkpoint molecules, immune tolerance molecules, and the like.

Suitable intracellular inhibitory domains may be any functional unit of a polypeptide as short as a 3 amino acid linear motif and as long as an entire protein, where size of the inhibitory domain is restricted only in that the domain must be sufficiently large as to retain its function and sufficiently small so as to be compatible with the other components of the polypeptide. Accordingly, an inhibitory domain may range in size from 3 amino acids in length to 1000 amino acids or more and, in some instances, can have a length of from about 30 amino acids to about 70 amino acids (aa), e.g., an inhibitory domain can have a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa. In other cases, an inhibitory domain can have a length of from about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.

In some instances, “co-inhibitory domains” find use in the subject polypeptides. Such co-inhibitory domains are generally polypeptides derived from receptors. Co-inhibition generally refers to the secondary inhibition of primary antigen-specific activation mechanisms which prevents co-stimulation. Co-inhibition, e.g., T cell co-inhibition, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42 and Thaventhiran et al. J Clin Cell Immunol (2012) S12, the disclosures of which are incorporated herein by reference in their entirety. In some embodiments, co-inhibitory domains homodimerize. In some instances, useful co-inhibitory domains have been modified to constitutively dimerize, including constitutively homodimerize. A subject co-inhibitory domain can be an intracellular portion of a transmembrane protein (i.e., the co-inhibitory domain can be derived from a transmembrane protein). Non-limiting examples of suitable co-inhibitory polypeptides include, but are not limited to, CTLA-4 and PD-1. In some instances, a co-inhibitory domain, e.g., as used in a subject polypeptide may include a co-inhibitory domain selected from PD-1, CTLA4, HPK1, SHP1, SHP2, Sts1 and Csk. In some instances, a co-inhibitory domain of subject polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a co-inhibitory domain as described herein.

In some instances, an antigen-binding inhibitory polypeptide may include a domain of a dimerization pair, such as e.g., a synthetic immune cell receptor (ICR) repressor useful as a component of a heteromeric, conditionally repressible synthetic ICR. Components of a heteromeric, conditionally repressible synthetic ICR may include a synthetic stimulatory ICR and a synthetic ICR repressor, where e.g., the synthetic stimulatory ICR and the synthetic ICR repressor specifically bind the antigens of an antigen pair described herein. Heteromeric, conditionally repressible synthetic ICRs, and components thereof, are described in PCT Application No. PCT/US2016/062612; the disclosure of which is incorporated herein by reference in its entirety.

Genetically Modified Immune Cells

The present disclosure provides a cytotoxic immune cell genetically modified to produce two antigen-triggered polypeptides, each recognizing a different cell surface antigen.

To generate a genetically modified cytotoxic immune cell of the present disclosure, a parent cytotoxic immune cell is genetically modified to produce: a) a first antigen-triggered polypeptide that binds specifically to a first target cell surface antigen present on a target cancer cell; and b) a second antigen-triggered polypeptide that binds specifically to a second target cell surface antigen. Suitable parent cytotoxic immune cells include CD8⁺ T cells, natural killer (NK) cells, and the like. Thus, in some cases, a genetically modified cytotoxic immune cell of the present disclosure is a genetically modified CD8⁺ T cell. In other cases, a genetically modified cytotoxic immune cell of the present disclosure is a genetically modified NK cell.

In some cases, the target cancer cell is a liposarcoma, a glioblastoma, a breast cancer cell, a renal cancer cell, a pancreatic cancer cell, a melanoma, an anaplastic lymphoma, a leiomyosarcoma, an astrocytoma, an ovarian cancer cell, a neuroblastoma, a mantle cell lymphoma, a sarcoma, a non-small cell lung cancer cell, an AML cell, a stomach cancer cell, a B-cell cancer cell, a lung cancer cell, or an oligodendroglioma.

In some cases, a genetically modified cytotoxic immune cell of the present disclosure is genetically modified to express a first antigen-triggered polypeptide and a second antigen-triggered polypeptide that bind to antigens of a 2-input AND-gate target antigen pair. In some cases, a genetically modified cytotoxic immune cell of the present disclosure is genetically modified to express a first antigen-triggered polypeptide and a second antigen-triggered polypeptide that bind to antigens of a 3-input AND-gate target antigen pair. Non-limiting examples of 2-input AND gates (AND gates based on 2 target antigens) and 3-input AND gates (AND gates based on 3 target antigens) are depicted schematically in FIG. 3A-3B.

For example, in some cases, the first antigen-triggered polypeptide is a synNotch receptor and activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces expression of the second antigen-triggered polypeptide. The second antigen-triggered polypeptide binds to the second antigen of the target antigen pair, where the second antigen is expressed on the surface of the target cancer cell. As an example, in some cases, the first antigen-triggered polypeptide is a synNotch receptor and the second antigen-triggered polypeptide is a single chain CAR. As another example, in some cases, the first antigen-triggered polypeptide is a synNotch receptor and the second antigen-triggered polypeptide is a TCR. For example, in some cases, the synNotch polypeptide comprises an intracellular domain comprising a transcriptional activator, and activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces release of the transcriptional activator; the released transcriptional activator activates transcription of the TCR or the single-chain CAR.

As another example, in some cases, the first antigen-triggered polypeptide is a synNotch receptor and activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces expression of the second antigen-triggered polypeptide, where the second antigen-triggered polypeptide is a heterodimeric (“two chain” or “split”) CAR comprising a first polypeptide chain and a second polypeptide chain. The heterodimeric CAR binds to the second antigen of the target antigen pair, where the second antigen is expressed on the surface of the target cancer cell. For example, in some cases, the first antigen-triggered polypeptide is a synNotch receptor and the second antigen-triggered polypeptide is a split CAR (e.g., an ON-switch CAR). In some cases, activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces expression of only the first polypeptide chain of the heterodimeric CAR; expression of the second polypeptide chain of the heterodimeric CAR can be constitutive. For example, in some cases, the synNotch polypeptide comprises an intracellular domain comprising a transcriptional activator, and activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces release of the transcriptional activator; the released transcriptional activator activates transcription of the first polypeptide chain of the heterodimeric CAR. In some cases, activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces expression of only the second polypeptide chain of the heterodimeric CAR; expression of the first polypeptide chain of the heterodimeric CAR can be constitutive. Once the first polypeptide chain of the heterodimeric CAR is produced in the cell, it heterodimerizes with the second polypeptide chain of the heterodimeric CAR. As another example, in some cases, the synNotch polypeptide comprises an intracellular domain comprising a transcriptional activator, and activation of the synNotch receptor by binding to the first antigen (present on a target cancer cell) induces release of the transcriptional activator; the released transcriptional activator activates transcription of the second polypeptide chain of the heterodimeric CAR.

In some cases, a genetically modified cytotoxic immune cell of the present disclosure is genetically modified to express a first antigen-triggered polypeptide and a second antigen-triggered polypeptide that bind to antigens of a 2-input AND-NOT-gate target antigen pair. In some cases, a genetically modified cytotoxic immune cell of the present disclosure is genetically modified to express a first antigen-triggered polypeptide and a second antigen-triggered polypeptide that bind to antigens of a 3-input AND-NOT-gate target antigen pair. Non-limiting examples of 2-input AND-NOT gates (AND-NOT gates based on 2 target antigens) and 3-input AND-NOT gates (AND-NOT gates based on 3 target antigens) are depicted schematically in FIG. 3C-3D.

As an example, in some cases, the first antigen-triggered polypeptide is a CAR, and the second antigen-triggered polypeptide is an iCAR. Binding of the iCAR to the second antigen (present on the surface of a non-cancerous cell, but not on the surface of a target cancer cell) of a target antigen pair inhibits T-cell activation mediated by activation of the CAR upon binding to the first antigen (present on the surface of the target cancer cell and on the surface of the non-cancerous cell) of the target antigen pair. As another example, in some cases, the first antigen-triggered polypeptide is a TCR, and the second antigen-triggered polypeptide is an iCAR. Binding of the iCAR to the second antigen (present on the surface of a non-cancerous cell, but not on the surface of a target cancer cell) of a target antigen pair blocks or reduces T-cell activation mediated by activation of the TCR upon binding to the first antigen (present on the surface of the target cancer cell and on the surface of the non-cancerous cell) of the target antigen pair.

As another example, in some cases, the first antigen-triggered polypeptide is a CAR, and the second antigen-triggered polypeptide is a synNotch polypeptide comprising an intracellular domain that, when released upon activation of the synNotch polypeptide by binding to the second target antigen, induces expression of an intracellular inhibitor that inhibits T-cell activation mediated by activation of the CAR upon binding to the first antigen (present on the surface of the target cancer cell and on the surface of the non-cancerous cell) of the target antigen pair. As another example, in some cases, the first antigen-triggered polypeptide is a TCR, and the second antigen-triggered polypeptide is a synNotch polypeptide comprising an intracellular domain that, when released upon activation of the synNotch polypeptide by binding to the second target antigen, induces expression of an intracellular inhibitor that inhibits T-cell activation mediated by activation of the TCR upon binding to the first antigen (present on the surface of the target cancer cell and on the surface of the non-cancerous cell) of the target antigen pair.

As another example, in some cases, the first antigen-triggered polypeptide is a CAR, and the second antigen-triggered polypeptide is a synNotch polypeptide comprising an intracellular domain that, when released upon activation of the synNotch polypeptide by binding to the second target antigen, induces expression of an extracellular inhibitor that inhibits T-cell activation mediated by activation of the CAR upon binding to the first antigen (present on the surface of the target cancer cell and on the surface of the non-cancerous cell) of the target antigen pair. As another example, in some cases, the first antigen-triggered polypeptide is a TCR, and the second antigen-triggered polypeptide is a synNotch polypeptide comprising an intracellular domain that, when released upon activation of the synNotch polypeptide by binding to the second target antigen, induces expression of an extracellular inhibitor that inhibits T-cell activation mediated by activation of the TCR upon binding to the first antigen (present on the surface of the target cancer cell and on the surface of the non-cancerous cell) of the target antigen pair.

In some cases, a genetically modified immune cell of the present disclosure provides for a 3-input AND, AND-NOT gate, comprising: 1) a first target antigen of an AND-NOT gate target antigen pair depicted in FIG. 1 or FIG. 9-14 ; 2) a second target antigen of the AND-NOT gate target antigen pair; and 3) a third target antigen, where the third target antigen is a target antigen depicted in FIG. 4 or Table 3, where the third target antigen is a target antigen for the same cancer cell type as the target antigen pair. In some cases, the 3-input AND, AND-NOT gate comprises: 1) the first target antigen of an AND-NOT gate target antigen pair depicted in FIG. 1 or FIG. 9-14 ; AND-NOT 2) the second target antigen of the AND-NOT gate target antigen pair; AND 3) the third target antigen. In some cases, the 3-input AND, AND-NOT gate comprises: 1) the second target antigen of an AND-NOT gate target antigen pair depicted in FIG. 1 or FIG. 9-14 ; AND-NOT 2) the first target antigen of the AND-NOT gate target antigen pair; AND 3) the third target antigen.

As one non-limiting example, where the target AND-NOT gate antigen pair is NLGN1 AND-NOT TNFRSF17 (for a glioma cell) (as shown in FIG. 1 ), a suitable third target antigen is EphA2 (as shown in FIG. 4 ).

Systems for Inhibiting Cancer Cells

The present disclosure provides a system for inhibiting or killing a target cancer cell. A system of the present disclosure comprises: a) a first antigen-triggered polypeptide that binds specifically to a first target antigen present on the target cancer cell, or a first nucleic acid comprising a nucleotide sequence encoding the first antigen-triggered polypeptide; and b) a second antigen-triggered polypeptide that binds specifically to a second target antigen, or a second nucleic acid comprising a nucleotide sequence encoding the second antigen-triggered polypeptide.

In some cases, the target cancer cell is a liposarcoma, a glioblastoma, a breast cancer cell, a renal cancer cell, a pancreatic cancer cell, a melanoma, an anaplastic lymphoma, a leiomyosarcoma, an astrocytoma, an ovarian cancer cell, a neuroblastoma, a mantle cell lymphoma, a sarcoma, a non-small cell lung cancer cell, an AML cell, a stomach cancer cell, a B-cell cancer cell, a lung cancer cell, or an oligodendroglioma.

In some cases, as noted above, a system of the present disclosure comprises: a) a first antigen-triggered polypeptide that binds specifically to a first target antigen present on a target cancer cell; and b) a second antigen-triggered polypeptide that binds specifically to a second target antigen. In these instances, the polypeptides per se are introduced into an immune cell (e.g., CD8⁺ T cells and/or NK cells obtained from an individual). Methods of introducing polypeptides into a cell are known in the art; and any known method can be used. For example, in some cases, the first and the second antigen-triggered polypeptides comprise a protein transduction domain (PTD) at the N-terminus or the C-terminus of the polypeptides.

In some cases, as noted above, a system of the present disclosure comprises: a) a first nucleic acid comprising a nucleotide sequence encoding a first antigen-triggered polypeptide that binds specifically to a first target antigen present on a target cancer cell; and b) a second nucleic acid comprising a nucleotide sequence encoding a second antigen-triggered polypeptide that binds specifically to a second target antigen. In some cases, the first and the second antigen-triggered polypeptides are encoded by nucleotide sequences on separate nucleic acids. In other cases, the first and the second antigen-triggered polypeptides are encoded by nucleotide sequences present in the same nucleic acid. In some cases, the nucleic acid is a recombinant expression vector. In some cases, a system of the present disclosure comprises: a) a first recombinant expression vector comprising a nucleotide sequence encoding a first antigen-triggered polypeptide that binds specifically to a first target antigen present on a target cancer cell; and b) a second recombinant expression vector comprising a nucleotide sequence encoding a second antigen-triggered polypeptide that binds specifically to a second target antigen. In some cases, the nucleotide sequences are operably linked to a constitutive promoter. In some cases, the nucleotide sequences are operably linked to a regulatable promoter (e.g., an inducible promoter, a reversible promoter, etc.). In some cases, the nucleotide sequences are operably linked to an immune cell promoter, e.g., a T-cell specific promoter. In some cases, a system of the present disclosure comprises a recombinant expression vector comprising nucleotide sequences encoding: a) a first antigen-triggered polypeptide that binds specifically to a first target antigen present on a target cancer cell; and b) a second antigen-triggered polypeptide that binds specifically to a second target antigen. In some cases, the nucleotide sequences are operably linked to a constitutive promoter. In some cases, the nucleotide sequences are operably linked to a regulatable promoter (e.g., an inducible promoter, a reversible promoter, etc.). In some cases, the nucleotide sequences are operably linked to an immune cell promoter, e.g., a T-cell specific promoter.

Suitable promoters include, but are not limited to; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; a metallothionein-I promoter; and various art-known promoters. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

In some instances, nucleic acids present in a system of the present disclosure include immune cell specific promoters that are expressed in one or more immune cell types, including but not limited to lymphocytes, hematopoietic stem cells and/or progeny thereof (i.e., immune cell progenitors), etc. Any convenient and appropriate promoter of an immune cell specific gene may find use in nucleic acids of the present disclosure. In some instances, an immune cell specific promoter of a nucleic acid present in a system of the present disclosure may be a T cell specific promoter. In some instances, an immune cell specific promoter of a nucleic acid present in a system of the present disclosure may be a light and/or heavy chain immunoglobulin gene promoter and may or may not include one or more related enhancer elements.

In some instances, an immune cell specific promoter of a nucleic acid present in a system of the present disclosure may be a promoter of a B29 gene promoter, a CD14 gene promoter, a CD43 gene promoter, a CD45 gene promoter, a CD68 gene promoter, a IFN-β gene promoter, a WASP gene promoter, a T-cell receptor β-chain gene promoter, a V9 γ (TRGV9) gene promoter, a V2 δ (TRDV2) gene promoter, and the like.

In some instances, an immune cell specific promoter present in a system of a nucleic acid of the present disclosure may be a viral promoter expressed in immune cells. As such, in some instances, viral promoters useful in nucleic acids present in a system of the present disclosure include viral promoters derived from immune cells viruses, including but not limited to, e.g., lentivirus promoters (e.g., HIV, SIV, FIV, EIAV, or Visna promoters) including e.g., LTR promoter, etc., Retroviridae promoters including, e.g., HTLV-I promoter, HTLV-II promoter, etc., and the like.

In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, a CD4 gene promoter can be used; see, e.g., Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739; and Marodon et al. (2003) Blood 101:3416. As another example, a CD8 gene promoter can be used. NK cell-specific expression can be achieved by use of an Ncr1 (p46) promoter; see, e.g., Eckelhart et al. (2011) Blood 117:1565.

Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable recombinant expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

Antigen Combinations

A system of the present disclosure targets antigen combinations, where the targeting provides for killing of a target cancer cell. A genetically modified immune cell of the present disclosure targets antigen combinations, where the targeting provides for killing of a target cancer cell. Antigen combinations may also reduce off-target effects and/or increase specificity for a target cancer cell, where e.g., an antigen combination includes one or more AND NOT combinations. Examples of target antigen combinations, and corresponding exemplary but non-limiting target cancer cells, are depicted in FIG. 1 and FIG. 9-14 . The following antigen combinations are exemplary, and not meant to be limiting.

Liposarcoma Antigen Target Combinations

In some cases, an antigen combination for targeting a liposarcoma cell comprises EVA1B AND NOT ITGA6. In some cases, the ITGA6 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 62 or 998. In some cases, the EVA1B polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4400.

In some cases, an antigen combination for targeting a liposarcoma cell comprises ADAM12 AND NOT TACSTD2. In some cases, the TACSTD2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2631. In some cases, the ADAM12 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2839 or 4815.

Glioblastoma Antigen Target Combinations

In some cases, an antigen combination for targeting a glioblastoma cell comprises PTPRZ1 AND FOLR2. In some cases, the FOLR2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 261, 1261, 1262 or 1263. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting a glioblastoma cell comprises PTPRZ1 AND IGF1R. In some cases, the IGF1R polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 308. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting a glioblastoma cell comprises PTPRZ1 AND NCAM1. In some cases, the NCAM1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 173, 949 or 6595. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting a glioblastoma cell comprises PTPRZ1 AND CD70. In some cases, the CD70 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2331. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting a glioblastoma cell comprises PTPRZ1 AND AXL. In some cases, the AXL polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2438 or 4847. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting a glioblastoma cell comprises PTPRZ1 AND NOT NAALAD2. In some cases, the NAALAD2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3289. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting a glioblastoma cell comprises PTPRZ1 AND NOT TMEM170B. In some cases, the TMEM170B polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1195. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting a glioblastoma cell comprises PTPRZ1 AND NOT PIGK. In some cases, the PIGK polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3293. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting a glioblastoma cell comprises PTPRZ1 AND CDH5. In some cases, the CDH5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2475. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting a glioblastoma cell comprises PTPRZ1 AND NOT 100507547?. In some cases, the 100507547? polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 7099. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

Additional examples of target antigen pairs for targeting a glioblastoma cell are provided in FIG. 1 and FIG. 9-14 .

Breast Adenocarcinoma Target Antigen Combinations

In some cases, an antigen combination for targeting a breast adenocarcinoma cell comprises CYB561 AND NOT SLC2A12. In some cases, the SLC2A12 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5933. In some cases, the CYB561 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 646, 647 or 2500.

In some cases, an antigen combination for targeting a breast adenocarcinoma cell comprises RHBDF1 AND NOT TMEM256. In some cases, the TMEM256 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6112. In some cases, the RHBDF1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4919.

In some cases, an antigen combination for targeting a breast adenocarcinoma cell comprises HSD3B7 AND NOT TMEM256. In some cases, the TMEM256 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6112. In some cases, the HSD3B7 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1560, 1699, 1700, 4387 or 5132.

In some cases, an antigen combination for targeting a breast adenocarcinoma cell comprises ADAM12 AND NOT TMEM256. In some cases, the TMEM256 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6112. In some cases, the ADAM12 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2839 or 4815.

In some cases, an antigen combination for targeting a breast adenocarcinoma cell comprises SLC5A6 AND NOT TMEM256. In some cases, the TMEM256 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6112. In some cases, the SLC5A6 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4767.

In some cases, an antigen combination for targeting a breast adenocarcinoma cell comprises GGT5 AND NOT TSPAN8. In some cases, the TSPAN8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3085. In some cases, the GGT5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1173, 1174 or 2968.

In some cases, an antigen combination for targeting a breast adenocarcinoma cell comprises ADAM12 AND NOT ADGRD1. In some cases, the ADGRD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6838. In some cases, the ADAM12 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2839 or 4815.

In some cases, an antigen combination for targeting a breast adenocarcinoma cell comprises LTBP3 AND NOT HBD. In some cases, the HBD polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 148. In some cases, the LTBP3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1421, 2072 or 4761.

In some cases, an antigen combination for targeting a breast adenocarcinoma cell comprises MARVELD3 AND NOT TMEM220. In some cases, the TMEM220 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 429. In some cases, the MARVELD3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 653 or 5518.

In some cases, an antigen combination for targeting a breast adenocarcinoma cell comprises RHBDF1 AND NOT SMIM5. In some cases, the SMIM5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2039. In some cases, the RHBDF1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4919.

Additional examples of target antigen pairs for targeting a breast adenocarcinoma cell are provided in FIG. 1 and FIG. 9-14 .

Glioma Target Antigen Combinations

In some cases, an antigen combination for targeting a glioma cell comprises NLGN1 AND NOT TNFRSF17. In some cases, the TNFRSF17 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2312. In some cases, the NLGN1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4016.

In some cases, an antigen combination for targeting a glioma cell comprises ITGAV AND NOT TNFRSF17. In some cases, the TNFRSF17 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2312. In some cases, the ITGAV polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1802, 1803 or 2579.

In some cases, an antigen combination for targeting a glioma cell comprises SYT11 AND TNFRSF17. In some cases, the TNFRSF17 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2312. In some cases, the SYT11 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6023.

In some cases, an antigen combination for targeting a glioma cell comprises SCRG1 AND TNFRSF17. In some cases, the TNFRSF17 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2312. In some cases, the SCRG1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3667.

In some cases, an antigen combination for targeting a glioma cell comprises AGPAT5 AND NOT TNFRSF17. In some cases, the TNFRSF17 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2312. In some cases, the AGPAT5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4429.

In some cases, an antigen combination for targeting a glioma cell comprises PTPRZ1 AND NOT TNFRSF17. In some cases, the TNFRSF17 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2312. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting a glioma cell comprises NRCAM AND NOT FOLH1. In some cases, the FOLH1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 631 or 3057. In some cases, the NRCAM polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 768, 769 or 3195.

In some cases, an antigen combination for targeting a glioma cell comprises PRAF2 AND NOT FOLH1. In some cases, the FOLH1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 631 or 3057. In some cases, the PRAF2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3649.

In some cases, an antigen combination for targeting a glioma cell comprises XPR1 AND FOLH1. In some cases, the FOLH1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 631 or 3057. In some cases, the XPR1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1546 or 3112.

In some cases, an antigen combination for targeting a glioma cell comprises SYT11 AND NOT FOLH1. In some cases, the FOLH1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 631 or 3057. In some cases, the SYT11 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6023.

Additional examples of target antigen pairs for targeting a glioma cell are provided in FIG. 1 and FIG. 9-14 .

Breast Carcinoma (e.g., Ductal Breast Carcinoma) Target Antigen Combinations

In some cases, an antigen combination for targeting a breast carcinoma cell comprises CACFD1 AND NOT TMED6. In some cases, the TMED6 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5889. In some cases, the CACFD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1566 or 4310.

In some cases, an antigen combination for targeting a breast carcinoma cell comprises CACFD1 AND NOT TMEM61. In some cases, the TMEM61 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6652. In some cases, the CACFD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1566 or 4310.

In some cases, an antigen combination for targeting a breast carcinoma cell comprises CACFD1 AND NOT CDHR2. In some cases, the CDHR2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2306 or 4321. In some cases, the CACFD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1566 or 4310.

In some cases, an antigen combination for targeting a breast carcinoma cell comprises CACFD1 AND NOT TMEM246. In some cases, the TMEM246 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5332. In some cases, the CACFD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1566 or 4310.

In some cases, an antigen combination for targeting a breast carcinoma cell comprises CACFD1 AND NOT SLC4A4. In some cases, the SLC4A4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1125, 1504 or 2885. In some cases, the CACFD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1566 or 4310.

In some cases, an antigen combination for targeting a breast carcinoma cell comprises C10orf35 AND NOT SLC4A4. In some cases, the SLC4A4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1125, 1504 or 2885. In some cases, the C10orf35 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5959.

In some cases, an antigen combination for targeting a breast carcinoma cell comprises RNF121 AND NOT PTCH1. In some cases, the PTCH1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 77, 1071, 1072, 1073, 1074, 1075 or 1076. In some cases, the RNF121 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4420.

Renal Carcinoma Target Antigen Combinations

In some cases, an antigen combination for targeting a renal carcinoma cell comprises CAV2 AND NOT SCARA5. In some cases, the SCARA5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6392. In some cases, the CAV2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2320 or 6759.

Pancreas Carcinoma (e.g., Pancreatic Ductal Carcinoma) Target Antigen Combinations

In some cases, an antigen combination for targeting a pancreas carcinoma cell comprises CTRB2 AND NOT CDH18. In some cases, the CDH18 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2192 or 3172. In some cases, the CTRB2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 701.

In some cases, an antigen combination for targeting a pancreas carcinoma cell comprises TM4SF4 AND NOT ABCC2. In some cases, the ABCC2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 117. In some cases, the TM4SF4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3086.

In some cases, an antigen combination for targeting a pancreas carcinoma cell comprises TM4SF4 AND NOT TMEM56. In some cases, the TMEM56 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6068. In some cases, the TM4SF4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3086.

In some cases, an antigen combination for targeting a pancreas carcinoma cell comprises TM4SF4 AND NOT CYP3A7. In some cases, the CYP3A7 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 239. In some cases, the TM4SF4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3086.

In some cases, an antigen combination for targeting a pancreas carcinoma cell comprises TM4SF4 AND NOT CYP2J2. In some cases, the CYP2J2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 243. In some cases, the TM4SF4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3086.

In some cases, an antigen combination for targeting a pancreas carcinoma cell comprises TM4SF4 AND NOT CLYBL. In some cases, the CLYBL polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6965. In some cases, the TM4SF4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3086.

In some cases, an antigen combination for targeting a pancreas carcinoma cell comprises TM4SF4 AND NOT SLC35D1. In some cases, the SLC35D1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4040. In some cases, the TM4SF4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3086.

In some cases, an antigen combination for targeting a pancreas carcinoma cell comprises TM4SF4 AND NOT G6PC. In some cases, the G6PC polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 45. In some cases, the TM4SF4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3086.

In some cases, an antigen combination for targeting a pancreas carcinoma cell comprises TM4SF4 AND GJA1. In some cases, the GJA1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 51. In some cases, the TM4SF4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3086.

In some cases, an antigen combination for targeting a pancreas carcinoma cell comprises CTRB2 AND NPTN. In some cases, the NPTN polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2004, 2005, 3774 or 4295. In some cases, the CTRB2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 701.

Additional examples of target antigen pairs for targeting a pancreas carcinoma cell are provided in FIG. 1 and FIG. 9-14 .

Melanoma Target Antigen Combinations

In some cases, an antigen combination for targeting a melanoma cell comprises DBI AND NOT FCN2. In some cases, the FCN2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2966 or 4122. In some cases, the DBI polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1006, 1007 and 4684.

In some cases, an antigen combination for targeting a melanoma cell comprises KIAA1549L AND DBI. In some cases, the KIAA1549L polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3718. In some cases, the DBI polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1006, 1007 and 4684.

In some cases, an antigen combination for targeting a melanoma cell comprises TMED3 AND DBI. In some cases, the TMED3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3691. In some cases, the DBI polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1006, 1007 and 4684.

In some cases, an antigen combination for targeting a melanoma cell comprises SFXN3 AND NOT DBI. In some cases, the SFXN3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5209. In some cases, the DBI polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1006, 1007 and 4684.

In some cases, an antigen combination for targeting a melanoma cell comprises GPR19 AND DBI. In some cases, the GPR19 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3446. In some cases, the DBI polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1006, 1007 and 4684.

In some cases, an antigen combination for targeting a melanoma cell comprises DBI AND NOT ITGA2B. In some cases, the ITGA2B polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 123. In some cases, the DBI polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1006, 1007 and 4684.

In some cases, an antigen combination for targeting a melanoma cell comprises DBI AND NOT VSTM1. In some cases, the VSTM1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6800. In some cases, the DBI polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1006, 1007 and 4684.

In some cases, an antigen combination for targeting a melanoma cell comprises DBI AND BNIP1. In some cases, the BNIP1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2317, 3842, 3843 or 3844. In some cases, the DBI polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1006, 1007 and 4684.

In some cases, an antigen combination for targeting a melanoma cell comprises DBI AND NOT CD300LB. In some cases, the CD300LB polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6404. In some cases, the DBI polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1006, 1007 and 4684.

In some cases, an antigen combination for targeting a melanoma cell comprises MANBAL AND DBI. In some cases, the MANBAL polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 425 or 4873. In some cases, the DBI polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1006, 1007 and 4684.

Additional examples of target antigen pairs for targeting a melanoma cell are provided in FIG. 1 and FIG. 9-14 .

Anaplastic Lymphoma (e.g., Anaplastic Large-Cell Lymphoma) Target Antigen Combinations

In some cases, an antigen combination for targeting an anaplastic lymphoma cell comprises TNFRSF8 AND NOT GPC3. In some cases, the GPC3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2089, 2090, 2091 or 3062. In some cases, the TNFRSF8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2323 or 6140.

In some cases, an antigen combination for targeting an anaplastic lymphoma cell comprises TNFRSF8 AND NOT L1CAM. In some cases, the L1CAM polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 126, 1743 or 4990. In some cases, the TNFRSF8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2323 or 6140.

In some cases, an antigen combination for targeting an anaplastic lymphoma cell comprises TNFRSF8 AND NOT SDC1. In some cases, the SDC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 496 or 2747. In some cases, the TNFRSF8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2323 or 6140.

In some cases, an antigen combination for targeting an anaplastic lymphoma cell comprises TNFRSF8 AND NOT TNFRSF10A. In some cases, the TNFRSF10A polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2911. In some cases, the TNFRSF8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2323 or 6140.

In some cases, an antigen combination for targeting an anaplastic lymphoma cell comprises TNFRSF8 AND NOT ERBB2. In some cases, the ERBB2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 474 or 3051. In some cases, the TNFRSF8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2323 or 6140.

In some cases, an antigen combination for targeting an anaplastic lymphoma cell comprises TNFRSF8 AND NOT IL11RA. In some cases, the IL11RA polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1701, 3066 or 5993. In some cases, the TNFRSF8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2323 or 6140.

In some cases, an antigen combination for targeting an anaplastic lymphoma cell comprises TNFRSF8 AND NOT SYT15. In some cases, the SYT15 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 5268 or 6605. In some cases, the TNFRSF8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2323 or 6140.

In some cases, an antigen combination for targeting an anaplastic lymphoma cell comprises TNFRSF8 AND NOT CYP4F2. In some cases, the CYP4F2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 988. In some cases, the TNFRSF8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2323 or 6140.

In some cases, an antigen combination for targeting an anaplastic lymphoma cell comprises TNFRSF8 AND NOT KCNK13. In some cases, the KCNK13 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4869. In some cases, the TNFRSF8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2323 or 6140.

In some cases, an antigen combination for targeting an anaplastic lymphoma cell comprises TNFRSF8 AND NOT NTN1. In some cases, the NTN1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3138. In some cases, the TNFRSF8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2323 or 6140.

Additional examples of target antigen pairs for targeting an anaplastic lymphoma cell are provided in FIG. 1 and FIG. 9-14 .

Leiomyosarcoma Target Antigen Combinations

In some cases, an antigen combination for targeting a leiomyosarcoma cell comprises VANGL1 AND NOT SGPP2. In some cases, the SGPP2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6046. In some cases, the VANGL1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5808.

In some cases, an antigen combination for targeting a leiomyosarcoma cell comprises VANGL1 AND NOT KRTCAP3. In some cases, the KRTCAP3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2225 or 6396. In some cases, the VANGL1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5808.

In some cases, an antigen combination for targeting a leiomyosarcoma cell comprises NEMP1 AND NOT SMIM5. In some cases, the SMIM5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2039. In some cases, the NEMP1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1451 or 4053.

In some cases, an antigen combination for targeting a leiomyosarcoma cell comprises ATP2A2 AND NOT CRB3. In some cases, the CRB3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5838 or 6403. In some cases, the ATP2A2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1561, 2432 or 6245.

In some cases, an antigen combination for targeting a leiomyosarcoma cell comprises VANGL1 AND NOT F11R. In some cases, the F11R polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4278. In some cases, the VANGL1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5808.

In some cases, an antigen combination for targeting a leiomyosarcoma cell comprises VANGL1 AND NOT RNF43. In some cases, the RNF43 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4337. In some cases, the VANGL1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5808.

In some cases, an antigen combination for targeting a leiomyosarcoma cell comprises VANGL1 AND NOT SCNN1A. In some cases, the SCNN1A polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 756, 1946 or 1947. In some cases, the VANGL1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5808.

In some cases, an antigen combination for targeting a leiomyosarcoma cell comprises VANGL1 AND NOT SMIM5. In some cases, the SMIM5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2039. In some cases, the VANGL1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5808.

In some cases, an antigen combination for targeting a leiomyosarcoma cell comprises VANGL1 AND NOT ERMP1. In some cases, the ERMP1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5098. In some cases, the VANGL1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5808.

In some cases, an antigen combination for targeting a leiomyosarcoma cell comprises VANGL1 AND NOT CRB3. In some cases, the CRB3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5838 or 6403. In some cases, the VANGL1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5808.

Additional examples of target antigen pairs for targeting a leiomyocarcoma cell are provided in FIG. 1 and FIG. 9-14 .

Astrocytoma Target Antigen Combinations

In some cases, an antigen combination for targeting an astrocytoma cell comprises PTPRZ1 AND CLCN5. In some cases, the CLCN5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 22, 1361 or 1362. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting an astrocytoma cell comprises PTPRZ1 AND ADGRE1. In some cases, the ADGRE1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2514. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting an astrocytoma cell comprises PTPRZ1 AND F2R. In some cases, the F2R polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2519. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting an astrocytoma cell comprises PTPRZ1 AND CYB561A3. In some cases, the CYB561A3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 646, 647 or 2500. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting an astrocytoma cell comprises PTPRZ1 AND IL27. In some cases, the IL27 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5969. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting an astrocytoma cell comprises PTPRZ1 AND NOT GHITM. In some cases, the GHITM polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3930. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting an astrocytoma cell comprises PTPRZ1 AND NOT PRRT3. In some cases, the PRRT3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 7014. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting an astrocytoma cell comprises PTPRZ1 AND HAS2. In some cases, the HAS2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3273. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting an astrocytoma cell comprises PTPRZ1 AND LCAT. In some cases, the LCAT polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 71. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

In some cases, an antigen combination for targeting an astrocytoma cell comprises PTPRZ1 AND NOT EPCAM. In some cases, the EPCAM polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2632. In some cases, the PTPRZ1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2726.

Additional examples of target antigen pairs for targeting an astrocytoma are provided in FIG. 1 and FIG. 9-14 .

Ovarian Cancer (e.g., Serous Cvstadenocarcinoma; Papillary Adenocarcinoma) Target Antigen Combinations

In some cases, an antigen combination for targeting an ovarian cancer cell (e.g., a cystadenocarcinoma) comprises SVOPL AND NOT ARL6IP5. In some cases, the ARL6IP5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3483. In some cases, the SVOPL polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1618 or 6423.

In some cases, an antigen combination for targeting an ovarian cancer cell comprises FAAH2 AND NOT ARL6IP5. In some cases, the ARL6IP5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3483. In some cases, the FAAH2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6408.

In some cases, an antigen combination for targeting an ovarian cancer cell comprises GAL3ST4 AND NOT ARL6IP5. In some cases, the ARL6IP5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3483. In some cases, the GAL3ST4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5061.

In some cases, an antigen combination for targeting an ovarian cancer cell comprises SYNE4 AND NOT FAXDC2. In some cases, the FAXDC2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 4206 or 5337. In some cases, the SYNE4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 814.

In some cases, an antigen combination for targeting an ovarian cancer cell comprises SYNE4 AND NOT OS9. In some cases, the OS9 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 650, 651, 652 or 3569. In some cases, the SYNE4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 814.

In some cases, an antigen combination for targeting an ovarian cancer cell comprises SVOPL AND NOT ANTXR2. In some cases, the ANTXR2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1876 or 5582. In some cases, the SVOPL polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1618 or 6423.

In some cases, an antigen combination for targeting an ovarian cancer cell comprises SVOPL AND NOT COMT. In some cases, the COMT polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 232, 1531, 1532 or 3683. In some cases, the SVOPL polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1618 or 6423.

In some cases, an antigen combination for targeting an ovarian cancer cell comprises SVOPL AND NOT SLC31A2. In some cases, the SLC31A2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2490. In some cases, the SVOPL polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1618 or 6423.

In some cases, an antigen combination for targeting an ovarian cancer cell comprises SVOPL AND NOT MFSD4. In some cases, the MFSD4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6617. In some cases, the SVOPL polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1618 or 6423.

In some cases, an antigen combination for targeting an ovarian cancer cell comprises SVOPL AND NOT FMO5. In some cases, the FMO5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1775, 1776 or 2379. In some cases, the SVOPL polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1618 or 6423.

Additional examples of target antigen pairs for targeting an ovarian cancer cell (e.g., a cystadenocarcinoma) are provided in FIG. 1 and FIG. 9-14 .

Neuroblastoma Target Antigen Combinations

In some cases, an antigen combination for targeting a neuroblastoma cell comprises MARCH11 AND SDC1. In some cases, the SDC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 496 or 2747. In some cases, the MARCH11 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1209.

In some cases, an antigen combination for targeting a neuroblastoma cell comprises SLC10A4 AND NOT SDC1. In some cases, the SDC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 496 or 2747. In some cases, the SLC10A4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6095.

In some cases, an antigen combination for targeting a neuroblastoma cell comprises ST8SIA2 AND NOT SDC1. In some cases, the SDC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 496 or 2747. In some cases, the ST8SIA2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3413.

In some cases, an antigen combination for targeting a neuroblastoma cell comprises VANGL2 AND NOT SDC1. In some cases, the SDC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 496 or 2747. In some cases, the VANGL2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4631.

In some cases, an antigen combination for targeting a neuroblastoma cell comprises DIABLO AND NOT SDC1. In some cases, the SDC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 496 or 2747. In some cases, the DIABLO polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 4591 or 5805.

In some cases, an antigen combination for targeting a neuroblastoma cell comprises SLC10A4 AND NOT WT1. In some cases, the WT1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 111, 5031, 5032 or 5033. In some cases, the SLC10A4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6095.

In some cases, an antigen combination for targeting a neuroblastoma cell comprises ST8SIA2 AND NOT WT1. In some cases, the WT1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 111, 5031, 5032 or 5033. In some cases, the ST8SIA2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3413.

In some cases, an antigen combination for targeting a neuroblastoma cell comprises ST8SIA2 AND NOT FAP. In some cases, the FAP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3055. In some cases, the ST8SIA2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3413.

In some cases, an antigen combination for targeting a neuroblastoma cell comprises SLC10A4 AND NOT MAGEA1. In some cases, the MAGEA1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3191. In some cases, the SLC10A4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6095.

In some cases, an antigen combination for targeting a neuroblastoma cell comprises MARCH11 AND NOT MET. In some cases, the MARCH11 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1209. In some cases, the MET polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 76 or 1332.

Additional examples of target antigen pairs for targeting a neuroblastoma cell are provided in FIG. 1 and FIG. 9-14 .

Mantle Cell Lymphoma Target Antigen Combinations

In some cases, an antigen combination for targeting a mantle-cell lymphoma cell comprises CLECL1 AND NOT EPHA3. In some cases, the EPHA3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 3237 or 6669. In some cases, the CLECL1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6267.

In some cases, an antigen combination for targeting a mantle-cell lymphoma cell comprises CELSR1 AND NOT ERBB2. In some cases, the ERBB2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 474 or 3051. In some cases, the CELSR1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3895.

In some cases, an antigen combination for targeting a mantle-cell lymphoma cell comprises QSOX2 AND NOT ERBB2. In some cases, the ERBB2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 474 or 3051. In some cases, the QSOX2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6621.

In some cases, an antigen combination for targeting a mantle-cell lymphoma cell comprises CLECL1 AND NOT CD160. In some cases, the CD160 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3624. In some cases, the CLECL1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6267.

In some cases, an antigen combination for targeting a mantle-cell lymphoma cell comprises CELSR1 AND NOT SDC1. In some cases, the SDC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 496 or 2747. In some cases, the CELSR1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3895.

In some cases, an antigen combination for targeting a mantle-cell lymphoma cell comprises TNFRSF13C AND NOT SDC1. In some cases, the SDC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 496 or 2747. In some cases, the TNFRSF13C polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5547.

In some cases, an antigen combination for targeting a mantle-cell lymphoma cell comprises CELSR1 AND NOT ERBB3. In some cases, the ERBB3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 476 or 2517. In some cases, the CELSR1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3895.

In some cases, an antigen combination for targeting a mantle-cell lymphoma cell comprises TNFRSF13C AND NOT FAP. In some cases, the FAP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3055. In some cases, the TNFRSF13C polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5547.

In some cases, an antigen combination for targeting a mantle-cell lymphoma cell comprises SNN AND NOT NCAM1. In some cases, the NCAM1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 173, 949 or 6595. In some cases, the SNN polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2843.

In some cases, an antigen combination for targeting a mantle-cell lymphoma cell comprises TNFRSF10A AND NOT NCAM1. In some cases, the NCAM1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 173, 949 or 6595. In some cases, the TNFRSF10A polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2911.

Additional examples of target antigen pairs for targeting a mantle cell lymphoma are provided in FIG. 1 and FIG. 9-14 .

Sarcoma Target Antigen Combinations

In some cases, an antigen combination for targeting a sarcoma cell comprises ADAM12 AND NOT ERBB4. In some cases, the ERBB4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 914 or 3238. In some cases, the ADAM12 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2839 or 4815.

In some cases, an antigen combination for targeting a sarcoma cell comprises AXL AND NOT ST6GALNAC2. In some cases, the ST6GALNAC2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3493. In some cases, the AXL polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2438 or 4847.

In some cases, an antigen combination for targeting a sarcoma cell comprises ADAM12 AND NOT ERBB3. In some cases, the ERBB3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 476 or 2517. In some cases, the ADAM12 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2839 or 4815.

In some cases, an antigen combination for targeting a sarcoma cell comprises ADAM12 AND NOT NAALAD2. In some cases, the NAALAD2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3289. In some cases, the ADAM12 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2839 or 4815.

In some cases, an antigen combination for targeting a sarcoma cell comprises TRAM2 AND NOT TMEM265. In some cases, the TMEM265 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 7113. In some cases, the TRAM2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3738.

In some cases, an antigen combination for targeting a sarcoma cell comprises ADAM12 AND NOT PDZK1IP1. In some cases, the PDZK1IP1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3359. In some cases, the ADAM12 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2839 or 4815.

In some cases, an antigen combination for targeting a sarcoma cell comprises TRAM2 AND NOT LPCAT3. In some cases, the LPCAT3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3362. In some cases, the TRAM2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3738.

In some cases, an antigen combination for targeting a sarcoma cell comprises CD163L1 AND NOT PRSS16. In some cases, the PRSS16 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3389. In some cases, the CD163L1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6416.

In some cases, an antigen combination for targeting a sarcoma cell comprises ADAM12 AND NOT PRSS16. In some cases, the PRSS16 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3389. In some cases, the ADAM12 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2839 or 4815.

In some cases, an antigen combination for targeting a sarcoma cell comprises TRAM2 AND NOT PRSS16. In some cases, the PRSS16 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3389. In some cases, the TRAM2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3738.

Additional examples of target antigen pairs for targeting a sarcoma cell are provided in FIG. 1 and FIG. 9-14 .

Non-Small Cell Lung Cancer Target Antigen Combinations

In some cases, an antigen combination for targeting a non-small cell lung cancer cell comprises FAP AND NOT LRRN4CL. In some cases, the LRRN4CL polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6935. In some cases, the FAP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3055.

In some cases, an antigen combination for targeting a non-small-cell lung cancer cell comprises FAP AND NOT GALNT16. In some cases, the GALNT16 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2226 or 4700. In some cases, the FAP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3055.

In some cases, an antigen combination for targeting a non-small-cell lung cancer cell comprises FAP AND NOT PTGFR. In some cases, the PTGFR polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs 346 or 804. In some cases, the FAP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3055.

In some cases, an antigen combination for targeting a non-small-cell lung cancer cell comprises FAP AND NOT LCAT. In some cases, the LCAT polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 71. In some cases, the FAP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3055.

In some cases, an antigen combination for targeting a non-small-cell lung cancer cell comprises FAP AND NOT SFRP1. In some cases, the SFRP1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2753. In some cases, the FAP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3055.

In some cases, an antigen combination for targeting a non-small-cell lung cancer cell comprises FAP AND NOT ITGA7. In some cases, the ITGA7 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1800, 1801 or 2576. In some cases, the FAP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3055.

In some cases, an antigen combination for targeting a non-small-cell lung cancer cell comprises FAP AND NOT ABCB1. In some cases, the ABCB1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 331. In some cases, the FAP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3055.

In some cases, an antigen combination for targeting a non-small-cell lung cancer cell comprises FAP AND NOT RECK. In some cases, the RECK polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4774. In some cases, the FAP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3055.

In some cases, an antigen combination for targeting a non-small-cell lung cancer cell comprises FAP AND NOT ENPP1. In some cases, the ENPP1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3456. In some cases, the FAP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3055.

In some cases, an antigen combination for targeting a non-small-cell lung cancer cell comprises FAP AND NOT TGFBR3. In some cases, the TGFBR3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2803. In some cases, the FAP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3055.

Additional examples of target antigen pairs for targeting a non-small-cell lung cancer cell are provided in FIG. 1 and FIG. 9-14 .

Acute Myeloid Leukemia (AML) Target Antigen Combinations

In some cases, an antigen combination for targeting an acute myeloid leukemia cell comprises SLC22A16 AND NOT SDC1. In some cases, the SDC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 496 or 2747. In some cases, the SLC22A16 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5452.

In some cases, an antigen combination for targeting an acute myeloid leukemia cell comprises FUT4 AND NOT SDC1. In some cases, the SDC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 496 or 2747. In some cases, the FUT4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2534.

In some cases, an antigen combination for targeting an acute myeloid leukemia cell comprises ELANE AND NOT SDC1. In some cases, the SDC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 496 or 2747. In some cases, the ELANE polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2513.

In some cases, an antigen combination for targeting an acute myeloid leukemia cell comprises MS4A3 AND NOT SLAMF7. In some cases, the SLAMF7 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4788. In some cases, the MS4A3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 721, 735 or 3443.

In some cases, an antigen combination for targeting an acute myeloid leukemia cell comprises TMC8 AND NOT SLAMF7. In some cases, the SLAMF7 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4788. In some cases, the TMC8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6065.

In some cases, an antigen combination for targeting an acute myeloid leukemia cell comprises NOT PFN2 AND NOT SLAMF7. In some cases, the SLAMF7 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4788. In some cases, the PFN2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2695 or 5556.

In some cases, an antigen combination for targeting an acute myeloid leukemia cell comprises CD33 AND NOT SLAMF7. In some cases, the SLAMF7 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4788. In some cases, the CD33 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1062 or 2462.

In some cases, an antigen combination for targeting an acute myeloid leukemia cell comprises FUT4 AND NOT SLAMF7. In some cases, the SLAMF7 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4788. In some cases, the FUT4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2534.

In some cases, an antigen combination for targeting an acute myeloid leukemia cell comprises NRROS AND NOT SLAMF7. In some cases, the SLAMF7 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4788. In some cases, the NRROS polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6817.

In some cases, an antigen combination for targeting an acute myeloid leukemia cell comprises FUT4 AND NOT SLAMF7. In some cases, the SLAMF7 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4788. In some cases, the FUT4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2534.

Additional examples of target antigen pairs for targeting an AML cell are provided in FIG. 1 and FIG. 9-14 .

Stomach Cancer Target Antigen Combinations

In some cases, an antigen combination for targeting a stomach cancer cell comprises MUC13 AND NOT SLC30A10. In some cases, the SLC30A10 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4487. In some cases, the MUC13 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5442.

In some cases, an antigen combination for targeting a stomach cancer cell comprises MUC13 AND NOT SCNN1B. In some cases, the SCNN1B polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 96. In some cases, the MUC13 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5442.

In some cases, an antigen combination for targeting a stomach cancer cell comprises MUC13 AND APOLD1. In some cases, the APOLD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1423 or 5181. In some cases, the MUC13 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5442.

In some cases, an antigen combination for targeting a stomach cancer cell comprises MUC13 AND GREM1. In some cases, the GREM1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3812. In some cases, the MUC13 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5442.

In some cases, an antigen combination for targeting a stomach cancer cell comprises MUC13 AND NOT DHRS9. In some cases, the DHRS9 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1628, 1629, 3364 or 6865. In some cases, the MUC13 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5442.

In some cases, an antigen combination for targeting a stomach cancer cell comprises MUC13 AND NOT TRPM6. In some cases, the TRPM6 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4319. In some cases, the MUC13 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5442.

In some cases, an antigen combination for targeting a stomach cancer cell comprises MUC13 AND NOT CLDN8. In some cases, the CLDN8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 6874. In some cases, the MUC13 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5442.

In some cases, an antigen combination for targeting a stomach cancer cell comprises MUC13 AND NOT SLC26A2. In some cases, the SLC26A2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 32. In some cases, the MUC13 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5442.

In some cases, an antigen combination for targeting a stomach cancer cell comprises MUC13 AND NOT CWH43. In some cases, the CWH43 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5119. In some cases, the MUC13 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 5442.

In some cases, an antigen combination for targeting a stomach cancer cell comprises GREM1 AND TNFRSF10B. In some cases, the TNFRSF10B polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2910 or 5994. In some cases, the GREM1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3812.

Additional examples of target antigen pairs for targeting a stomach cancer cell are provided in FIG. 1 and FIG. 9-14 .

Diffuse Large B-Cell Lymphoma Target Antigen Combinations

In some cases, an antigen combination for targeting a diffuse large B-cell lymphoma cell comprises PLA2G2D AND NOT GABRD. In some cases, the GABRD polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 271. In some cases, the PLA2G2D polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3768.

In some cases, an antigen combination for targeting a diffuse large B-cell lymphoma cell comprises PLA2G2D AND NOT BMPR1B. In some cases, the BMPR1B polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2315. In some cases, the PLA2G2D polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3768.

In some cases, an antigen combination for targeting a diffuse large B-cell lymphoma cell comprises PLA2G2D AND NOT CRYGC. In some cases, the CRYGC polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4754. In some cases, the PLA2G2D polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3768/

In some cases, an antigen combination for targeting a diffuse large B-cell lymphoma cell comprises PLA2G2D AND NOT HCN4. In some cases, the HCN4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3292. In some cases, the PLA2G2D polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3768.

In some cases, an antigen combination for targeting a diffuse large B-cell lymphoma cell comprises PLA2G2D AND NOT SLC17A4. In some cases, the SLC17A4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3294. In some cases, the PLA2G2D polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3768.

In some cases, an antigen combination for targeting a diffuse large B-cell lymphoma cell comprises PLA2G2D AND NOT CDH7. In some cases, the CDH7 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 3031 or 5510. In some cases, the PLA2G2D polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3768.

In some cases, an antigen combination for targeting a diffuse large B-cell lymphoma cell comprises PLA2G2D AND NOT CDH12. In some cases, the CDH12 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2955. In some cases, the PLA2G2D polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3768.

In some cases, an antigen combination for targeting a diffuse large B-cell lymphoma cell comprises PLA2G2D AND NOT CLEC3A. In some cases, the CLEC3A polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3356. In some cases, the PLA2G2D polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3768.

In some cases, an antigen combination for targeting a diffuse large B-cell lymphoma cell comprises PLA2G2D AND NOT PDZK1IP1. In some cases, the PDZK1IP1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3359. In some cases, the PLA2G2D polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3768.

In some cases, an antigen combination for targeting a diffuse large B-cell lymphoma cell comprises PLA2G2D AND NOT TENM1. In some cases, the TENM1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2049, 2050 or 3896. In some cases, the PLA2G2D polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3768.

Additional examples of target antigen pairs for targeting a diffuse large B-cell lymphoma are provided in FIG. 1 and FIG. 9-14 .

Lung Cancer (e.g., Lung Adenocarcinoma) Target Antigen Combinations

In some cases, an antigen combination for targeting a lung cancer cell comprises SFTPD AND NOT IL3RA. In some cases, the SFTPD polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2755. In some cases, the IL3RA polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2567.

In some cases, an antigen combination for targeting a lung cancer cell comprises SFTPD AND NOT CDH5. In some cases, the SFTPD polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2755. In some cases, the CDH5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2475.

In some cases, an antigen combination for targeting a lung cancer cell comprises SFTPC AND NOT RAMP2. In some cases, the SFTPC polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2754. In some cases, the RAMP2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3385.

In some cases, an antigen combination for targeting a lung cancer cell comprises SFTPD AND NOT RAMP2. In some cases, the SFTPD polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2755. In some cases, the RAMP2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3385.

In some cases, an antigen combination for targeting a lung cancer cell comprises SFTPD AND NOT SIRPB1. In some cases, the SFTPD polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2755. In some cases, the SIRPB1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1082, 1573 or 3426.

In some cases, an antigen combination for targeting a lung cancer cell comprises SFTPD AND NOT ABCA8. In some cases, the SFTPD polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2755. In some cases, the ABCA8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3640.

In some cases, an antigen combination for targeting a lung cancer cell comprises SLC34A2 AND NOT DLL1. In some cases, the DLL1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3326. In some cases, the SLC34A2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3490.

In some cases, an antigen combination for targeting a lung cancer cell comprises SFTPC AND NOT LYVE1. In some cases, the LYVE1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3547. In some cases, the SFTPC polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2754.

In some cases, an antigen combination for targeting a lung cancer cell comprises SFTPD AND NOT LYVE1. In some cases, the SFTPD polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2755. In some cases, the LYVE1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3547.

In some cases, an antigen combination for targeting a lung cancer cell comprises SFTPD AND NOT PNPLA6. In some cases, the SFTPD polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2755. In some cases, the PNPLA6 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2146, 2147, 2148, 2149 or 3551.

Additional examples of target antigen pairs for targeting a lung cancer cell are provided in FIG. 1 and FIG. 9-14 .

Oligodendroglioma Target Antigen Combinations

In some cases, an antigen combination for targeting an oligodendroglioma cell comprises NLGN3 AND NOT FOLR2. In some cases, the FOLR2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 261, 1261, 1262 or 1263. In some cases, the NLGN3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2174, 4543 or 6589.

In some cases, an antigen combination for targeting an oligodendroglioma cell comprises NLGN3 AND NOT SLAMF7. In some cases, the SLAMF7 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4788. In some cases, the NLGN3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2174, 4543 or 6589.

In some cases, an antigen combination for targeting an oligodendroglioma cell comprises NLGN3 AND NOT SDC1. In some cases, the SDC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 496 or 2747. In some cases, the NLGN3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2174, 4543 or 6589.

In some cases, an antigen combination for targeting an oligodendroglioma cell comprises NLGN3 AND NOT TNFRSF8. In some cases, the TNFRSF8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2323 or 6140. In some cases, the NLGN3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2174, 4543 or 6589.

In some cases, an antigen combination for targeting an oligodendroglioma cell comprises NLGN3 AND NOT IL1IRA. In some cases, the IL1IRA polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1701, 3066 or 5993. In some cases, the NLGN3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2174, 4543 or 6589.

In some cases, an antigen combination for targeting an oligodendroglioma cell comprises NLGN3 AND NOT MUC1. In some cases, the MUC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 661, 662, 922, 923, 924, 925 or 2657. In some cases, the NLGN3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2174, 4543 or 6589.

In some cases, an antigen combination for targeting an oligodendroglioma cell comprises NLGN3 AND NOT KDR. In some cases, the KDR polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2607. In some cases, the NLGN3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2174, 4543 or 6589.

In some cases, an antigen combination for targeting an oligodendroglioma cell comprises NLGN3 AND NOT IGF1R. In some cases, the IGF1R polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 308. In some cases, the NLGN3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2174, 4543 or 6589.

In some cases, an antigen combination for targeting an oligodendroglioma cell comprises NLGN3 AND NOT ALK. In some cases, the ALK polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3013. In some cases, the NLGN3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2174, 4543 or 6589.

In some cases, an antigen combination for targeting an oligodendroglioma cell comprises NLGN3 AND NOT ERBB3. In some cases, the ERBB3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 476 or 2517. In some cases, the NLGN3 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2174, 4543 or 6589.

Additional examples of target antigen pairs for targeting an oligodendroglioma cell are provided in FIG. 1 and FIG. 9-14 .

Other Antigen Combinations

In some cases, an antigen combination may be selected for targeting a cancer where two antigens of the combination are “clinical antigens” (i.e., the antigen has been evaluated clinically (i.e., in an immunotherapy clinical trial) and found to be a marker for one or more cancers). An antigen combination having two or more clinical antigens may include any clinical antigen including those described herein.

Combinations of clinical antigens include combinations where an antigen is highly expressed (H) in a target cancer, where an antigen is lowly expressed (L) in a target cancer, where two antigens are highly expressed (HH) in a target cancer, where two antigens are lowly expressed (LL) in a target cancer, where a first antigen is highly expressed and the second antigen is lowly expressed (HL) in a target cancer, where a first antigen is lowly expressed and the second antigen is highly expressed (LH) in a target cancer, where two antigens are expressed (i.e., an “OR” gate) in a target cancer.

For example, in some instances, the following clinical-clinical antigen pairs may be employed in a subject antigen combination, alone or in combination with other antigens, where the pair is listed with one or more non-limiting exemplary cancers and an exemplary expression or gating structure as defined above: MLANA:PMEL (Melanoma; H), MLANA:B4GALNT1 (Melanoma; OR), MLANA:KDR (Melanoma; HL), EPCAM:MET (Breast; HL), MSLN:EPCAM (Breast; HL), IL3RA:CD33 (Leukemia, Myeloid, Acute; HH), EPCAM:TYR (Breast; HL), EPCAM:ROR1 (Breast; HH), CD19:MS4A1 (Lymphoma, Large B-Cell, Diffuse; OR), ERBB2:ROR1 (Breast; HL), ERBB2:TYR (Breast; HL), ERBB2:EPCAM (Breast; HH), MSLN:ERBB2 (Lung Adenocarcinoma; HL), MSLN:MUC1 (Lung Adenocarcinoma; H), ERBB2:MUC1 (Lung Adenocarcinoma; HL), ERBB2:MET (Breast; HL), MSLN:GPC3 (Colon; LH), EPHA2:ERBB2 (Glioma; LH), EPHA2:MUC1 (Glioma; LH), MUC1:ROR1 (Lung Carcinoma; LH), GPC3:MUC1 (Colon; LH), ERBB2:GPC3 (Colon; LH), CD19:CD22 (Lymphoma, Large B-Cell, Diffuse; HL), ROR1:TYR (Breast; LH), MS4A1:CD22 (Lymphoma, Large B-Cell, Diffuse; HL), MSLN:ERBB2 (Carcinoma, Pancreatic Ductal; HL), MET:ROR1 (Breast; LH), MET:TYR (Breast; LH), MSLN:ROR1 (Breast; HL), MSLN:TYR (Breast; HL), MUC1:TNFRSF10A (Lung Carcinoma; OR), MSLN:MET (Breast; HL), ROR1:TNFRSF10A (Lung Carcinoma; LH), MSLN:ERBB2 (Lung Carcinoma; HL), MSLN:GPC3 (Lung Carcinoma; LH), MSLN:MUC1 (Lung Carcinoma; LH), MSLN:ROR1 (Lung Carcinoma; LH), MSLN:TYR (Lung Carcinoma; LH), MSLN:TNFRSF10A (Lung Carcinoma; LH), ERBB2:GPC3 (Lung Carcinoma; -), ERBB2:MUC1 (Lung Carcinoma; LH), ERBB2:ROR1 (Lung Carcinoma; LH), GPC3:MUC1 (Lung Carcinoma; LH), GPC3:ROR1 (Lung Carcinoma; LH), GPC3:TYR (Lung Carcinoma; LH), GPC3:TNFRSF10A (Lung Carcinoma; LH), MUC1:TYR (Lung Carcinoma; LH), ROR1:TYR (Lung Carcinoma; LH), TYR:TNFRSF10A (Lung Carcinoma; LH), MSLN:ERBB2 (Ovarian Neoplasms, Cystadenocarcinoma, Adenocarcinoma; HL), Flt3:CD123 (acute myeloid leukemia; HH), Flt3:CD33 (acute myeloid leukemia; HH) and GPC3:AFP (hepatocellular carcinoma; HH).

In some cases, useful clinical-clinical antigen pairs may include Flt3:CD123, for example as an AND-gate. In some instances, Flt3:CD123 may find use in targeting acute myeloid leukemia. In some cases, useful clinical-clinical antigen pairs may include Flt3:CD33, for example as an AND-gate. In some instances, Flt3:CD33 may find use in targeting acute myeloid leukemia. In some cases, useful clinical-clinical antigen pairs may include GPC3:AFP, for example as an AND-gate. In some instances, GPC3:AFP may find use in targeting hepatocellular carcinoma.

In some cases, an antigen combination may include one or more antigens of interest, where an “antigen of interest” may include clinical antigens as well as antigens that are of clinical interest for reasons other than having been identified as associated with a particular cancer type. Such other reasons include but are not limited to e.g., association with cancer stem cells (i.e., a cancer stem cell marker). Antigens of interest that may be included in antigen combinations include, e.g., those listed in Table 3 below.

TABLE 3 ALK ULBP2 CLDN1 (SEQ ID (SEQ ID (SEQ ID NO: 3013) NO: 5133) NO: 4771) AXL ULBP3 CLDN10 (SEQ ID (SEQ ID (SEQ ID NO: 2438) NO: 7298) NO: 1975) CD276 RAET1E CLDN11 (SEQ ID (SEQ ID (SEQ ID NO: 685) NO: 5840) NO: 3322) NCR3LG1 RAET1G CLDN12 (SEQ ID (SEQ ID (SEQ ID NO: 7428) NO: 7369) NO: 3711) TNFRSF17 RAET1L CLDN14 (SEQ ID (SEQ ID (SEQ ID NO: 2312) NO: 7368) NO: 1902) CA9 CD34 CLDN15 (SEQ ID (SEQ ID (SEQ ID NO: 2318) NO: 697) NO: 3920) IL3RA ALDH1A1 CLDN16 (SEQ ID (SEQ ID (SEQ ID NO: 2567) NO: 7313) NO: 3520) SDC1 PROMI CLDN17 (SEQ ID (SEQ ID (SEQ ID NO: 496) NO: 1878) NO: 3713) CD160 FLOT2 CLDN18 (SEQ ID (SEQ ID (SEQ ID NO: 3624) NO: 7359) NO: 387) L1CAM CD24 CLDN19 (SEQ ID (SEQ ID (SEQ ID NO: 126) NO: 7446) NO: 1296) CD19 CBX3 CLDN2 (SEQ ID (SEQ ID (SEQ ID NO: 2460) NO: 7304) NO: 2267) MS4A1 ABCA5 CLDN20 (SEQ ID (SEQ ID (SEQ ID NO: 4853) NO: 4483) NO: 7334) CD22 ABCB5 CLDN24 (SEQ ID (SEQ ID (SEQ ID NO: 2461) NO: 7412) NO: 7422) TNFRSF8 IGB1 CLDN22 (SEQ ID (SEQ ID (SEQ ID NO: 2323) NO: 7309) NO: 7381) CD33 LGR5 CLDN23 (SEQ ID (SEQ ID (SEQ ID NO: 1062) NO: 2866) NO: 6713) CD38 ALCAM CLDN3 (SEQ ID (SEQ ID (SEQ ID NO: 2465) NO: 2420) NO: 2343) CD44 THY1 CLDN4 (SEQ ID (SEQ ID (SEQ ID NO: 172) NO: 7454) NO: 2342) CD70 DNAJB8 CLDN5 (SEQ ID (SEQ ID (SEQ ID NO: 2331) NO: 7318) NO: 1443) SLAMF7 DDX3X CLDN6 (SEQ ID (SEQ ID (SEQ ID NO: 4788) NO: 7424) NO: 4792) EPCAM DLL3 CLDN7 (SEQ ID (SEQ ID (SEQ ID NO: 2632) NO: 4275) NO: 2344) EPHA2 BMPR1B CLDN8 (SEQ ID (SEQ ID (SEQ ID NO: 3042) NO: 2315) NO: 6874) EPHA3 SLC7A5 CLDN9 (SEQ ID (SEQ ID (SEQ ID NO: 3237) NO: 7352) NO: 4752) ERBB3 STEAP1 CLEC14A (SEQ ID (SEQ ID (SEQ ID NO: 476) NO: 3777) NO: 6440) ERBB4 IL13RA1 CTAG1A (SEQ ID (SEQ ID (SEQ ID NO: 914) NO: 2415) NO: 7312) FAP SLC34A2 CTAG2 (SEQ ID (SEQ ID (SEQ ID NO: 3055) NO: 3490) NO: 7442) FOLR1 SEMA5B DKKI (SEQ ID (SEQ ID (SEQ ID NO: 260) NO: 7357) NO: 7282) FOLR2 GPR37L1 DPEP1 (SEQ ID (SEQ ID (SEQ ID NO: 261) NO: 3118) NO: 7391) B4GALNT1 RNF43 EDNRB (SEQ ID (SEQ ID (SEQ ID NO: 2385) NO: 4337) NO: 33) ST8SIA1 STEAP2 EGFR (SEQ ID (SEQ ID (SEQ ID NO: 2758) NO: 879) NO: 3235) GPA33 TRPM4 ENG (SEQ ID (SEQ ID (SEQ ID NO: 3374) NO: 4315) NO: 35) GPC3 TDGF1 ENPP3 (SEQ ID (SEQ ID (SEQ ID NO: 2089) NO: 7419) NO: 3199) ERBB2 CR2 GAGE1 (SEQ ID (SEQ ID (SEQ ID NO: 474) NO: 493) NO: 7360) CTAG1B CD79B GPNMB (SEQ ID (SEQ ID (SEQ ID NO: 7258) NO: 178) NO: 454) CSPG4 FCRL2 GUCY2C (SEQ ID (SEQ ID (SEQ ID NO: 2497) NO: 5166) NO: 3179) IGF1R CEACAM6 HAVCR1 (SEQ ID (SEQ ID (SEQ ID NO: 308) NO: 7445) NO: 7417) ILIIRA IL20RA HHLA2 (SEQ ID (SEQ ID (SEQ ID NO: 1701) NO: 3936) NO: 3626) IL13RA2 BCAN HSPA5 (SEQ ID (SEQ ID (SEQ ID NO: 185) NO: 7332) NO: 7305) IGK EPHB2 IL2RA (SEQ ID (SEQ ID (SEQ ID NO: 7467) NO: 3047) NO: 7271) MAGEA1 TNFRSF13C ITGAV (SEQ ID (SEQ ID (SEQ ID NO: 3191) NO: 5547) NO: 1802) MAGEA3 CD74 ITGB6 (SEQ ID (SEQ ID (SEQ ID NO: 7279) NO: 7351) NO: 313) MLANA CD79A MAGEA11 (SEQ ID (SEQ ID (SEQ ID NO: 3302) NO: 7257) NO: 7346) MSLN CXCR5 MAGEA4 (SEQ ID (SEQ ID (SEQ ID NO: 3377) NO: 7256) NO: 7341) MET HLA-DOB MOK (SEQ ID (SEQ ID (SEQ ID NO: 76) NO: 2555) NO: 7436) MUC13 P2RX5 MST1R (SEQ ID (SEQ ID (SEQ ID NO: 5442) NO: 2680) NO: 2656) MUC17 CD72 MUC4 (SEQ ID (SEQ ID (SEQ ID NO: 835) NO: 2470) NO: 3070) MUCI CD180 0AS1 (SEQ ID (SEQ ID (SEQ ID NO: 661) NO: 3318) NO: 7354) MUC16 FCRL1 PCYT1A (SEQ ID (SEQ ID (SEQ ID NO: 5067) NO: 1941) NO: 7455) NCAMI FCRL5 PMEL (SEQ ID (SEQ ID (SEQ ID NO: 173) NO: 5213) NO: 3597) PSCA TMEFF2 PRR4 (SEQ ID (SEQ ID (SEQ ID NO: 3340) NO: 4183) NO: 7429) FOLH1 SST PTK7 (SEQ ID (SEQ ID (SEQ ID NO: 631) NO: 7266) NO: 2709) TNFSF11 SSTR2 SLC39A6 (SEQ ID (SEQ ID (SEQ ID NO: 2871) NO: 928) NO: 1158) ROR1 SSTR5 SPON2 (SEQ ID (SEQ ID (SEQ ID NO: 1070) NO: 937) NO: 7392) TNFRSF10A SSTR1 SSTR4 (SEQ ID (SEQ ID (SEQ ID NO:2911) NO: 927) NO: 936) TNFRSF10B SSTR3 SSX1 (SEQ ID (SEQ ID (SEQ ID NO: 2910) NO: 935) NO: 7438) KDR AFP TDGF1P3 (SEQ ID (SEQ ID (SEQ ID NO: 2607) NO: 7252) NO: 7466) WTI AGER TNC (SEQ ID (SEQ ID (SEQ ID NO: 111) NO: 1470) NO: 2562) ITGB1 ANXA1 TPBG (SEQ ID (SEQ ID (SEQ ID NO: 7310) NO: 7253) NO: 2164) ITGB3 BAGE TYR (SEQ ID (SEQ ID (SEQ ID NO: 64) NO: 7269) NO: 110) FUT3 BIRC5 VCAM1 (SEQ ID (SEQ ID (SEQ ID NO: 43) NO: 7342) NO: 946) CEACAM5 CD37 VTCN1 (SEQ ID (SEQ ID (SEQ ID NO: 7444) NO: 823) NO: 5057) ULBP1 CD52 (SEQ ID (SEQ ID NO: 7458) NO: 2478)

In some cases, useful antigen combinations may include an antigen combination that includes a clinical antigen in combination with a tissue antigen (e.g., an antigen expressed in normal brain tissue) in an AND NOT gate. For example, useful antigen combinations may include a GD2 (B4GALNT1; SEQ ID NO:2385) clinical antigen AND NOT a tissue antigen selected from: OPALIN (SEQ ID NO:833), TMEM235 (SEQ ID NO:1869), GABRA1 (SEQ ID NO:262), KCNJ9 (SEQ ID NO:3190), GRM3 (SEQ ID NO:290), SEZ6 (SEQ ID NO:1143), NTSR2 (SEQ ID NO:3753), KCNK4 (SEQ ID NO:5486), SLCO1A2 (SEQ ID NO:7290), SLC24A2 (SEQ ID NO:4632), MOG (SEQ ID NO:528), GABRG1 (SEQ ID NO:6354), GABRG2 (SEQ ID NO:272), CNTNAP4 (SEQ ID NO:5492), DSCAM (SEQ ID NO:2354), CACNG3 (SEQ ID NO:3510), CRB1 (SEQ ID NO:6891), CDH10 (SEQ ID NO:3552), HRH3 (SEQ ID NO:3657), GRIK1 (SEQ ID NO:280), SLC39A12 (SEQ ID NO:1826), GPR158 (SEQ ID NO:4716), CACNG2 (SEQ ID NO:3430), SYT3 (SEQ ID NO:7410), HTR5A (SEQ ID NO:4993), CACNG7 (SEQ ID NO:5264), GPR37L1 (SEQ ID NO:3118), LRRTM3 (SEQ ID NO:7362), GLRA2 (SEQ ID NO:1281), CHRNB2 (SEQ ID NO:228), KCNQ2 (SEQ ID NO:3067), JPH3 (SEQ ID NO:4690), GPR19 (SEQ ID NO:3446), ADCY8 (SEQ ID NO:1248), SPOCK3 (SEQ ID NO:845), SLC32A1 (SEQ ID NO:5606), OPCML (SEQ ID NO:594), GABRA3 (SEQ ID NO:264), GRM5 (SEQ ID NO:292), SCN1A (SEQ ID NO:2133), SLC5A11 (SEQ ID NO:5546), KCNC1 (SEQ ID NO:1249), SLC12A5 (SEQ ID NO:1506), GRM4 (SEQ ID NO:291), GRM1 (SEQ ID NO:288), GRIA4 (SEQ ID NO:279), MEGF11 (SEQ ID NO:5354), CACNA1B (SEQ ID NO:211), LYPD1 (SEQ ID NO:970), GRID2 (SEQ ID NO:2398), SCN2A (SEQ ID NO:842), NKAIN2 (SEQ ID NO:864), UNC5A (SEQ ID NO:5690), SLC4A10 (SEQ ID NO:7421), TMEFF2 (SEQ ID NO:4183), CSMD3 (SEQ ID NO:5532), PPAPDC1A (SEQ ID NO:715), HAPLN4 (SEQ ID NO:4968), GPR85 (SEQ ID NO:1920), ANTXR2 (SEQ ID NO:1876), CACNG4 (SEQ ID NO:3934), CSPG5 (SEQ ID NO:3516), KCNK10 (SEQ ID NO:4784), CHRNA4 (SEQ ID NO:225), CNTNAP2 (SEQ ID NO:3871), KCNJ10 (SEQ ID NO:2597), GABRB2 (SEQ ID NO:269), GRIN1 (SEQ ID NO:282), CRB2 (SEQ ID NO:6381), SHISA7 (SEQ ID NO:7406), NKAIN4 (SEQ ID NO:6122), HTR2C (SEQ ID NO:301), CACNG8 (SEQ ID NO:5263), NRG3 (SEQ ID NO:569), ABCG4 (SEQ ID NO:1668), CDH8 (SEQ ID NO:2476), GABRD (SEQ ID NO:271), KIRREL3 (SEQ ID NO:2022), GABRB1 (SEQ ID NO:268), KCNA2 (SEQ ID NO:3181), CDH20 (SEQ ID NO:5261), IGDCC3 (SEQ ID NO:3159), KCNJ6 (SEQ ID NO:2596), CNIH2 (SEQ ID NO:6658), KCNK12 (SEQ ID NO:7295), CDH18 (SEQ ID NO:2192), CSMD2 (SEQ ID NO:5530), SYT4 (SEQ ID NO:4722), OR2L13 (SEQ ID NO:6468), CDH9 (SEQ ID NO:4198), GABRA2 (SEQ ID NO:263), KCNF1 (SEQ ID NO:2592), MAG (SEQ ID NO:2634), CALN1 (SEQ ID NO:645), GRIN2B (SEQ ID NO:284), GRM7 (SEQ ID NO:294), VSTM2A (SEQ ID NO:7459), GPR61 (SEQ ID NO:5271), OMG (SEQ ID NO:2672), KCNA1 (SEQ ID NO:66), GPR83 (SEQ ID NO:4239), ATP8A2 (SEQ ID NO:4237), GABBR2 (SEQ ID NO:3288), GPR12 (SEQ ID NO:3255), TRPM3 (SEQ ID NO:519), SLC8A3 (SEQ ID NO:1424), KCND2 (SEQ ID NO:7288), GSG1L (SEQ ID NO:1236), SLC30A10 (SEQ ID NO:4487), ASTN1 (SEQ ID NO:3016), GPR179 (SEQ ID NO:7447), LRFN2 (SEQ ID NO:4711), CACNA1E (SEQ ID NO:214), CALY (SEQ ID NO:4119), SLC6A15 (SEQ ID NO:1932), KIAA0319 (SEQ ID NO:2227), SYT6 (SEQ ID NO:6957), PTPRR (SEQ ID NO:2724), KCTD8 (SEQ ID NO:7331), GPR22 (SEQ ID NO:3261), SLC4A8 (SEQ ID NO:818), LAMP5 (SEQ ID NO:3729), MEGF10 (SEQ ID NO:5355), FXYD7 (SEQ ID NO:4864), KCNK9 (SEQ ID NO:4257), SLC1A6 (SEQ ID NO:3206), MLC1 (SEQ ID NO:4042), OPRK1 (SEQ ID NO:325), ATP2B2 (SEQ ID NO:353), ACSL6 (SEQ ID NO:560), THBD (SEQ ID NO:108), PTPRT (SEQ ID NO:3622), PCDHGC4 (SEQ ID NO:2688), CLDN10 (SEQ ID NO:1975), KCNV1 (SEQ ID NO:3925), LPPR3 (SEQ ID NO:5096), SLCO1C1 (SEQ ID NO:1885), PCDH8 (SEQ ID NO:2690), ANO4 (SEQ ID NO:6566), LRRTM4 (SEQ ID NO:1505), PCDH15 (SEQ ID NO:1688), CCKBR (SEQ ID NO:6481), GABRA5 (SEQ ID NO:266), SLC6A12 (SEQ ID NO:1290), GRIN2A (SEQ ID NO:283), SLC1A2 (SEQ ID NO:2977), SLC43A1 (SEQ ID NO:2856), KCNC2 (SEQ ID NO:5836), ELFN2 (SEQ ID NO:5534), ATP7A (SEQ ID NO:10), GRIK4 (SEQ ID NO:3965), LRRC55 (SEQ ID NO:448), HCN2 (SEQ ID NO:7377), NKAIN1 (SEQ ID NO:5039), DPP10 (SEQ ID NO:432), AJAP1 (SEQ ID NO:894), NPFFR1 (SEQ ID NO:4886), TRPC3 (SEQ ID NO:1434), TGFBI (SEQ ID NO:107), SLC6A7 (SEQ ID NO:7370), GABRA4 (SEQ ID NO:265), SLC13A5 (SEQ ID NO:1732), GRIN2C (SEQ ID NO:285), HCN1 (SEQ ID NO:4763), SLC26A8 (SEQ ID NO:5552), PPIC (SEQ ID NO:336), NETO1 (SEQ ID NO:7427), TNFRSF10B (SEQ ID NO:2910), CDH22 (SEQ ID NO:4803), SLC6A13 (SEQ ID NO:4264), DISP2 (SEQ ID NO:7320), SLC6A11 (SEQ ID NO:3890), CD93 (SEQ ID NO:3698), EPHA10 (SEQ ID NO:1160), PHLDB2 (SEQ ID NO:1490), OXTR (SEQ ID NO:7326), WNT7A (SEQ ID NO:3089), GYPC (SEQ ID NO:7432), KCNA4 (SEQ ID NO:2589), PCDHAC2 (SEQ ID NO:4498), HGFAC (SEQ ID NO:2405), DRD1 (SEQ ID NO:253), SHISA9 (SEQ ID NO:7418), SCN8A (SEQ ID NO:3879), ICAM1 (SEQ ID NO:7382), PIRT (SEQ ID NO:1201), A4GALT (SEQ ID NO:4291), MRGPRF (SEQ ID NO:1129), CD248 (SEQ ID NO:4649), CD58 (SEQ ID NO:7405), CD44 (SEQ ID NO:172), EPHA2 (SEQ ID NO:3042) or PROCR (SEQ ID NO:3480). In some instances, such antigen combinations may be useful in targeting melanoma.

Useful antigen combinations may include a MAGEA1 (SEQ ID NO:3191) clinical antigen AND NOT a tissue antigen (e.g., a brain tissue antigen) selected from: OPALIN (SEQ ID NO:833), TMEM235 (SEQ ID NO:1869), GABRA1 (SEQ ID NO:262), KCNJ9 (SEQ ID NO:3190), GRM3 (SEQ ID NO:290), SEZ6 (SEQ ID NO:1143), NTSR2 (SEQ ID NO:3753), KCNK4 (SEQ ID NO:5486), SLCO1A2 (SEQ ID NO:7290), SLC24A2 (SEQ ID NO:4632), MOG (SEQ ID NO:528), GABRG1 (SEQ ID NO:6354), GABRG2 (SEQ ID NO:272), CNTNAP4 (SEQ ID NO:5492), DSCAM (SEQ ID NO:2354), CACNG3 (SEQ ID NO:3510), CRB1 (SEQ ID NO:6891), CDH10 (SEQ ID NO:3552), HRH3 (SEQ ID NO:3657), GRIK1 (SEQ ID NO:280), SLC39A12 (SEQ ID NO:1826), GPR158 (SEQ ID NO:4716), CACNG2 (SEQ ID NO:3430), SYT3 (SEQ ID NO:7410), HTR5A (SEQ ID NO:4993), CACNG7 (SEQ ID NO:5264), GPR37L1 (SEQ ID NO:3118), LRRTM3 (SEQ ID NO:7362), GLRA2 (SEQ ID NO:1281), CHRNB2 (SEQ ID NO:228), KCNQ2 (SEQ ID NO:3067), JPH3 (SEQ ID NO:4690), GPR19 (SEQ ID NO:3446), ADCY8 (SEQ ID NO:1248), SPOCK3 (SEQ ID NO:845), SLC32A1 (SEQ ID NO:5606), OPCML (SEQ ID NO:594), GABRA3 (SEQ ID NO:264), GRM5 (SEQ ID NO:292), SCN1A (SEQ ID NO:2133), SLC5A11 (SEQ ID NO:5546), KCNC1 (SEQ ID NO:1249), SLC12A5 (SEQ ID NO:1506), GRM4 (SEQ ID NO:291), GRM1 (SEQ ID NO:288), GRIA4 (SEQ ID NO:279), MEGF11 (SEQ ID NO:5354), CACNA1B (SEQ ID NO:211), LYPD1 (SEQ ID NO:970), GRID2 (SEQ ID NO:2398), SCN2A (SEQ ID NO:842), NKAIN2 (SEQ ID NO:864), UNC5A (SEQ ID NO:5690), SLC4A10 (SEQ ID NO:7421), TMEFF2 (SEQ ID NO:4183), CSMD3 (SEQ ID NO:5532), PPAPDC1A (SEQ ID NO:715), HAPLN4 (SEQ ID NO:4968), GPR85 (SEQ ID NO:1920), ANTXR2 (SEQ ID NO:1876), CACNG4 (SEQ ID NO:3934), CSPG5 (SEQ ID NO:3516), KCNK10 (SEQ ID NO:4784), CHRNA4 (SEQ ID NO:225), CNTNAP2 (SEQ ID NO:3871), KCNJ10 (SEQ ID NO:2597), GABRB2 (SEQ ID NO:269), GRIN1 (SEQ ID NO:282), CRB2 (SEQ ID NO:6381), SHISA7 (SEQ ID NO:7406), NKAIN4 (SEQ ID NO:6122), HTR2C (SEQ ID NO:301), CACNG8 (SEQ ID NO:5263), NRG3 (SEQ ID NO:569), ABCG4 (SEQ ID NO:1668), CDH8 (SEQ ID NO:2476), GABRD (SEQ ID NO:271), KIRREL3 (SEQ ID NO:2022), GABRB1 (SEQ ID NO:268), KCNA2 (SEQ ID NO:3181), CDH20 (SEQ ID NO:5261), IGDCC3 (SEQ ID NO:3159), KCNJ6 (SEQ ID NO:2596), CNIH2 (SEQ ID NO:6658), KCNK12 (SEQ ID NO:7295), CDH18 (SEQ ID NO:2192), CSMD2 (SEQ ID NO:5530), SYT4 (SEQ ID NO:4722), OR2L13 (SEQ ID NO:6468), CDH9 (SEQ ID NO:4198), GABRA2 (SEQ ID NO:263), KCNF1 (SEQ ID NO:2592), MAG (SEQ ID NO:2634), CALN1 (SEQ ID NO:645), GRIN2B (SEQ ID NO:284), GRM7 (SEQ ID NO:294), VSTM2A (SEQ ID NO:7459), GPR61 (SEQ ID NO:5271), OMG (SEQ ID NO:2672), KCNA1 (SEQ ID NO:66), GPR83 (SEQ ID NO:4239), ATP8A2 (SEQ ID NO:4237), GABBR2 (SEQ ID NO:3288), GPR12 (SEQ ID NO:3255), TRPM3 (SEQ ID NO:519), SLC8A3 (SEQ ID NO:1424), KCND2 (SEQ ID NO:7288), GSG1L (SEQ ID NO:1236), SLC30A10 (SEQ ID NO:4487), ASTN1 (SEQ ID NO:3016), GPR179 (SEQ ID NO:7447), LRFN2 (SEQ ID NO:4711), CACNA1E (SEQ ID NO:214), CALY (SEQ ID NO:4119), SLC6A15 (SEQ ID NO:1932), KIAA0319 (SEQ ID NO:2227), SYT6 (SEQ ID NO:6957), PTPRR (SEQ ID NO:2724), KCTD8 (SEQ ID NO:7331), GPR22 (SEQ ID NO:3261), SLC4A8 (SEQ ID NO:818), LAMP5 (SEQ ID NO:3729), MEGF10 (SEQ ID NO:5355), FXYD7 (SEQ ID NO:4864), KCNK9 (SEQ ID NO:4257), SLC1A6 (SEQ ID NO:3206), MLC1 (SEQ ID NO:4042), OPRK1 (SEQ ID NO:325), ATP2B2 (SEQ ID NO:353), ACSL6 (SEQ ID NO:560), THBD (SEQ ID NO:108), PTPRT (SEQ ID NO:3622), PCDHGC4 (SEQ ID NO:2688), CLDN10 (SEQ ID NO:1975), KCNV1 (SEQ ID NO:3925), LPPR3 (SEQ ID NO:5096), SLCO1C1 (SEQ ID NO:1885), PCDH8 (SEQ ID NO:2690), ANO4 (SEQ ID NO:6566), LRRTM4 (SEQ ID NO:1505), PCDH15 (SEQ ID NO:1688), CCKBR (SEQ ID NO:6481), GABRA5 (SEQ ID NO:266), SLC6A12 (SEQ ID NO:1290), GRIN2A (SEQ ID NO:283), SLC1A2 (SEQ ID NO:2977), SLC43A1 (SEQ ID NO:2856), KCNC2 (SEQ ID NO:5836), ELFN2 (SEQ ID NO:5534), ATP7A (SEQ ID NO:10), GRIK4 (SEQ ID NO:3965), LRRC55 (SEQ ID NO:448), HCN2 (SEQ ID NO:7377), NKAIN1 (SEQ ID NO:5039), DPP10 (SEQ ID NO:432), AJAP1 (SEQ ID NO:894), NPFFR1 (SEQ ID NO:4886), TRPC3 (SEQ ID NO:1434), TGFBI (SEQ ID NO:107), SLC6A7 (SEQ ID NO:7370), GABRA4 (SEQ ID NO:265), SLC13A5 (SEQ ID NO:1732), GRIN2C (SEQ ID NO:285), HCN1 (SEQ ID NO:4763), SLC26A8 (SEQ ID NO:5552), PPIC (SEQ ID NO:336), NETO1 (SEQ ID NO:7427), TNFRSF10B (SEQ ID NO:2910), CDH22 (SEQ ID NO:4803), SLC6A13 (SEQ ID NO:4264), DISP2 (SEQ ID NO:7320), SLC6A11 (SEQ ID NO:3890), CD93 (SEQ ID NO:3698), EPHA10 (SEQ ID NO:1160), PHLDB2 (SEQ ID NO:1490), OXTR (SEQ ID NO:7326), WNT7A (SEQ ID NO:3089), GYPC (SEQ ID NO:7432), KCNA4 (SEQ ID NO:2589), PCDHAC2 (SEQ ID NO:4498), HGFAC (SEQ ID NO:2405), DRD1 (SEQ ID NO:253), SHISA9 (SEQ ID NO:7418), SCN8A (SEQ ID NO:3879), ICAM1 (SEQ ID NO:7382), PIRT (SEQ ID NO:1201), A4GALT (SEQ ID NO:4291), MRGPRF (SEQ ID NO:1129), CD248 (SEQ ID NO:4649), CD58 (SEQ ID NO:7405), CD44 (SEQ ID NO:172), EPHA2 (SEQ ID NO:3042) or PROCR (SEQ ID NO:3480). In some instances, such antigen combinations may be useful in targeting melanoma.

Useful antigen combinations may include a MAGEA3 (SEQ ID NO:7279) clinical antigen AND NOT a tissue antigen (e.g., a brain tissue antigen) selected from: OPALIN (SEQ ID NO:833), TMEM235 (SEQ ID NO:1869), GABRA1 (SEQ ID NO:262), KCNJ9 (SEQ ID NO:3190), GRM3 (SEQ ID NO:290), SEZ6 (SEQ ID NO:1143), NTSR2 (SEQ ID NO:3753), KCNK4 (SEQ ID NO:5486), SLCO1A2 (SEQ ID NO:7290), SLC24A2 (SEQ ID NO:4632), MOG (SEQ ID NO:528), GABRG1 (SEQ ID NO:6354), GABRG2 (SEQ ID NO:272), CNTNAP4 (SEQ ID NO:5492), DSCAM (SEQ ID NO:2354), CACNG3 (SEQ ID NO:3510), CRB1 (SEQ ID NO:6891), CDH10 (SEQ ID NO:3552), HRH3 (SEQ ID NO:3657), GRIK1 (SEQ ID NO:280), SLC39A12 (SEQ ID NO:1826), GPR158 (SEQ ID NO:4716), CACNG2 (SEQ ID NO:3430), SYT3 (SEQ ID NO:7410), HTR5A (SEQ ID NO:4993), CACNG7 (SEQ ID NO:5264), GPR37L1 (SEQ ID NO:3118), LRRTM3 (SEQ ID NO:7362), GLRA2 (SEQ ID NO:1281), CHRNB2 (SEQ ID NO:228), KCNQ2 (SEQ ID NO:3067), JPH3 (SEQ ID NO:4690), GPR19 (SEQ ID NO:3446), ADCY8 (SEQ ID NO:1248), SPOCK3 (SEQ ID NO:845), SLC32A1 (SEQ ID NO:5606), OPCML (SEQ ID NO:594), GABRA3 (SEQ ID NO:264), GRM5 (SEQ ID NO:292), SCN1A (SEQ ID NO:2133), SLC5A11 (SEQ ID NO:5546), KCNC1 (SEQ ID NO:1249), SLC12A5 (SEQ ID NO:1506), GRM4 (SEQ ID NO:291), GRM1 (SEQ ID NO:288), GRIA4 (SEQ ID NO:279), MEGF11 (SEQ ID NO:5354), CACNA1B (SEQ ID NO:211), LYPD1 (SEQ ID NO:970), GRID2 (SEQ ID NO:2398), SCN2A (SEQ ID NO:842), NKAIN2 (SEQ ID NO:864), UNC5A (SEQ ID NO:5690), SLC4A10 (SEQ ID NO:7421), TMEFF2 (SEQ ID NO:4183), CSMD3 (SEQ ID NO:5532), PPAPDC1A (SEQ ID NO:715), HAPLN4 (SEQ ID NO:4968), GPR85 (SEQ ID NO:1920), ANTXR2 (SEQ ID NO:1876), CACNG4 (SEQ ID NO:3934), CSPG5 (SEQ ID NO:3516), KCNK10 (SEQ ID NO:4784), CHRNA4 (SEQ ID NO:225), CNTNAP2 (SEQ ID NO:3871), KCNJ10 (SEQ ID NO:2597), GABRB2 (SEQ ID NO:269), GRIN1 (SEQ ID NO:282), CRB2 (SEQ ID NO:6381), SHISA7 (SEQ ID NO:7406), NKAIN4 (SEQ ID NO:6122), HTR2C (SEQ ID NO:301), CACNG8 (SEQ ID NO:5263), NRG3 (SEQ ID NO:569), ABCG4 (SEQ ID NO:1668), CDH8 (SEQ ID NO:2476), GABRD (SEQ ID NO:271), KIRREL3 (SEQ ID NO:2022), GABRB1 (SEQ ID NO:268), KCNA2 (SEQ ID NO:3181), CDH20 (SEQ ID NO:5261), IGDCC3 (SEQ ID NO:3159), KCNJ6 (SEQ ID NO:2596), CNIH2 (SEQ ID NO:6658), KCNK12 (SEQ ID NO:7295), CDH18 (SEQ ID NO:2192), CSMD2 (SEQ ID NO:5530), SYT4 (SEQ ID NO:4722), OR2L13 (SEQ ID NO:6468), CDH9 (SEQ ID NO:4198), GABRA2 (SEQ ID NO:263), KCNF1 (SEQ ID NO:2592), MAG (SEQ ID NO:2634), CALN1 (SEQ ID NO:645), GRIN2B (SEQ ID NO:284), GRM7 (SEQ ID NO:294), VSTM2A (SEQ ID NO:7459), GPR61 (SEQ ID NO:5271), OMG (SEQ ID NO:2672), KCNA1 (SEQ ID NO:66), GPR83 (SEQ ID NO:4239), ATP8A2 (SEQ ID NO:4237), GABBR2 (SEQ ID NO:3288), GPR12 (SEQ ID NO:3255), TRPM3 (SEQ ID NO:519), SLC8A3 (SEQ ID NO:1424), KCND2 (SEQ ID NO:7288), GSG1L (SEQ ID NO:1236), SLC30A10 (SEQ ID NO:4487), ASTN1 (SEQ ID NO:3016), GPR179 (SEQ ID NO:7447), LRFN2 (SEQ ID NO:4711), CACNA1E (SEQ ID NO:214), CALY (SEQ ID NO:4119), SLC6A15 (SEQ ID NO:1932), KIAA0319 (SEQ ID NO:2227), SYT6 (SEQ ID NO:6957), PTPRR (SEQ ID NO:2724), KCTD8 (SEQ ID NO:7331), GPR22 (SEQ ID NO:3261), SLC4A8 (SEQ ID NO:818), LAMP5 (SEQ ID NO:3729), MEGF10 (SEQ ID NO:5355), FXYD7 (SEQ ID NO:4864), KCNK9 (SEQ ID NO:4257), SLC1A6 (SEQ ID NO:3206), MLC1 (SEQ ID NO:4042), OPRK1 (SEQ ID NO:325), ATP2B2 (SEQ ID NO:353), ACSL6 (SEQ ID NO:560), THBD (SEQ ID NO:108), PTPRT (SEQ ID NO:3622), PCDHGC4 (SEQ ID NO:2688), CLDN10 (SEQ ID NO:1975), KCNV1 (SEQ ID NO:3925), LPPR3 (SEQ ID NO:5096), SLCO1C1 (SEQ ID NO:1885), PCDH8 (SEQ ID NO:2690), ANO4 (SEQ ID NO:6566), LRRTM4 (SEQ ID NO:1505), PCDH15 (SEQ ID NO:1688), CCKBR (SEQ ID NO:6481), GABRA5 (SEQ ID NO:266), SLC6A12 (SEQ ID NO:1290), GRIN2A (SEQ ID NO:283), SLC1A2 (SEQ ID NO:2977), SLC43A1 (SEQ ID NO:2856), KCNC2 (SEQ ID NO:5836), ELFN2 (SEQ ID NO:5534), ATP7A (SEQ ID NO:10), GRIK4 (SEQ ID NO:3965), LRRC55 (SEQ ID NO:448), HCN2 (SEQ ID NO:7377), NKAIN1 (SEQ ID NO:5039), DPP10 (SEQ ID NO:432), AJAP1 (SEQ ID NO:894), NPFFR1 (SEQ ID NO:4886), TRPC3 (SEQ ID NO:1434), TGFBI (SEQ ID NO:107), SLC6A7 (SEQ ID NO:7370), GABRA4 (SEQ ID NO:265), SLC13A5 (SEQ ID NO:1732), GRIN2C (SEQ ID NO:285), HCN1 (SEQ ID NO:4763), SLC26A8 (SEQ ID NO:5552), PPIC (SEQ ID NO:336), NETO1 (SEQ ID NO:7427), TNFRSF10B (SEQ ID NO:2910), CDH22 (SEQ ID NO:4803), SLC6A13 (SEQ ID NO:4264), DISP2 (SEQ ID NO:7320), SLC6A11 (SEQ ID NO:3890), CD93 (SEQ ID NO:3698), EPHA10 (SEQ ID NO:1160), PHLDB2 (SEQ ID NO:1490), OXTR (SEQ ID NO:7326), WNT7A (SEQ ID NO:3089), GYPC (SEQ ID NO:7432), KCNA4 (SEQ ID NO:2589), PCDHAC2 (SEQ ID NO:4498), HGFAC (SEQ ID NO:2405), DRD1 (SEQ ID NO:253), SHISA9 (SEQ ID NO:7418), SCN8A (SEQ ID NO:3879), ICAM1 (SEQ ID NO:7382), PIRT (SEQ ID NO:1201), A4GALT (SEQ ID NO:4291), MRGPRF (SEQ ID NO:1129), CD248 (SEQ ID NO:4649), CD58 (SEQ ID NO:7405), CD44 (SEQ ID NO:172), EPHA2 (SEQ ID NO:3042) or PROCR (SEQ ID NO:3480). In some instances, such antigen combinations may be useful in targeting melanoma.

Useful antigen combinations may include a MART1 (MLANA; SEQ ID NO:3302) clinical antigen AND NOT a tissue antigen (e.g., a brain tissue antigen) selected from: OPALIN (SEQ ID NO:833), TMEM235 (SEQ ID NO:1869), GABRA1 (SEQ ID NO:262), KCNJ9 (SEQ ID NO:3190), GRM3 (SEQ ID NO:290), SEZ6 (SEQ ID NO:1143), NTSR2 (SEQ ID NO:3753), KCNK4 (SEQ ID NO:5486), SLCO1A2 (SEQ ID NO:7290), SLC24A2 (SEQ ID NO:4632), MOG (SEQ ID NO:528), GABRG1 (SEQ ID NO:6354), GABRG2 (SEQ ID NO:272), CNTNAP4 (SEQ ID NO:5492), DSCAM (SEQ ID NO:2354), CACNG3 (SEQ ID NO:3510), CRB1 (SEQ ID NO:6891), CDH10 (SEQ ID NO:3552), HRH3 (SEQ ID NO:3657), GRIK1 (SEQ ID NO:280), SLC39A12 (SEQ ID NO:1826), GPR158 (SEQ ID NO:4716), CACNG2 (SEQ ID NO:3430), SYT3 (SEQ ID NO:7410), HTR5A (SEQ ID NO:4993), CACNG7 (SEQ ID NO:5264), GPR37L1 (SEQ ID NO:3118), LRRTM3 (SEQ ID NO:7362), GLRA2 (SEQ ID NO:1281), CHRNB2 (SEQ ID NO:228), KCNQ2 (SEQ ID NO:3067), JPH3 (SEQ ID NO:4690), GPR19 (SEQ ID NO:3446), ADCY8 (SEQ ID NO:1248), SPOCK3 (SEQ ID NO:845), SLC32A1 (SEQ ID NO:5606), OPCML (SEQ ID NO:594), GABRA3 (SEQ ID NO:264), GRM5 (SEQ ID NO:292), SCN1A (SEQ ID NO:2133), SLC5A11 (SEQ ID NO:5546), KCNC1 (SEQ ID NO:1249), SLC12A5 (SEQ ID NO:1506), GRM4 (SEQ ID NO:291), GRM1 (SEQ ID NO:288), GRIA4 (SEQ ID NO:279), MEGF11 (SEQ ID NO:5354), CACNA1B (SEQ ID NO:211), LYPD1 (SEQ ID NO:970), GRID2 (SEQ ID NO:2398), SCN2A (SEQ ID NO:842), NKAIN2 (SEQ ID NO:864), UNC5A (SEQ ID NO:5690), SLC4A10 (SEQ ID NO:7421), TMEFF2 (SEQ ID NO:4183), CSMD3 (SEQ ID NO:5532), PPAPDC1A (SEQ ID NO:715), HAPLN4 (SEQ ID NO:4968), GPR85 (SEQ ID NO:1920), ANTXR2 (SEQ ID NO:1876), CACNG4 (SEQ ID NO:3934), CSPG5 (SEQ ID NO:3516), KCNK10 (SEQ ID NO:4784), CHRNA4 (SEQ ID NO:225), CNTNAP2 (SEQ ID NO:3871), KCNJ10 (SEQ ID NO:2597), GABRB2 (SEQ ID NO:269), GRIN1 (SEQ ID NO:282), CRB2 (SEQ ID NO:6381), SHISA7 (SEQ ID NO:7406), NKAIN4 (SEQ ID NO:6122), HTR2C (SEQ ID NO:301), CACNG8 (SEQ ID NO:5263), NRG3 (SEQ ID NO:569), ABCG4 (SEQ ID NO:1668), CDH8 (SEQ ID NO:2476), GABRD (SEQ ID NO:271), KIRREL3 (SEQ ID NO:2022), GABRB1 (SEQ ID NO:268), KCNA2 (SEQ ID NO:3181), CDH20 (SEQ ID NO:5261), IGDCC3 (SEQ ID NO:3159), KCNJ6 (SEQ ID NO:2596), CNIH2 (SEQ ID NO:6658), KCNK12 (SEQ ID NO:7295), CDH18 (SEQ ID NO:2192), CSMD2 (SEQ ID NO:5530), SYT4 (SEQ ID NO:4722), OR2L13 (SEQ ID NO:6468), CDH9 (SEQ ID NO:4198), GABRA2 (SEQ ID NO:263), KCNF1 (SEQ ID NO:2592), MAG (SEQ ID NO:2634), CALN1 (SEQ ID NO:645), GRIN2B (SEQ ID NO:284), GRM7 (SEQ ID NO:294), VSTM2A (SEQ ID NO:7459), GPR61 (SEQ ID NO:5271), OMG (SEQ ID NO:2672), KCNA1 (SEQ ID NO:66), GPR83 (SEQ ID NO:4239), ATP8A2 (SEQ ID NO:4237), GABBR2 (SEQ ID NO:3288), GPR12 (SEQ ID NO:3255), TRPM3 (SEQ ID NO:519), SLC8A3 (SEQ ID NO:1424), KCND2 (SEQ ID NO:7288), GSG1L (SEQ ID NO:1236), SLC30A10 (SEQ ID NO:4487), ASTN1 (SEQ ID NO:3016), GPR179 (SEQ ID NO:7447), LRFN2 (SEQ ID NO:4711), CACNA1E (SEQ ID NO:214), CALY (SEQ ID NO:4119), SLC6A15 (SEQ ID NO:1932), KIAA0319 (SEQ ID NO:2227), SYT6 (SEQ ID NO:6957), PTPRR (SEQ ID NO:2724), KCTD8 (SEQ ID NO:7331), GPR22 (SEQ ID NO:3261), SLC4A8 (SEQ ID NO:818), LAMP5 (SEQ ID NO:3729), MEGF10 (SEQ ID NO:5355), FXYD7 (SEQ ID NO:4864), KCNK9 (SEQ ID NO:4257), SLC1A6 (SEQ ID NO:3206), MLC1 (SEQ ID NO:4042), OPRK1 (SEQ ID NO:325), ATP2B2 (SEQ ID NO:353), ACSL6 (SEQ ID NO:560), THBD (SEQ ID NO:108), PTPRT (SEQ ID NO:3622), PCDHGC4 (SEQ ID NO:2688), CLDN10 (SEQ ID NO:1975), KCNV1 (SEQ ID NO:3925), LPPR3 (SEQ ID NO:5096), SLCO1C1 (SEQ ID NO:1885), PCDH8 (SEQ ID NO:2690), ANO4 (SEQ ID NO:6566), LRRTM4 (SEQ ID NO:1505), PCDH15 (SEQ ID NO:1688), CCKBR (SEQ ID NO:6481), GABRA5 (SEQ ID NO:266), SLC6A12 (SEQ ID NO:1290), GRIN2A (SEQ ID NO:283), SLC1A2 (SEQ ID NO:2977), SLC43A1 (SEQ ID NO:2856), KCNC2 (SEQ ID NO:5836), ELFN2 (SEQ ID NO:5534), ATP7A (SEQ ID NO:10), GRIK4 (SEQ ID NO:3965), LRRC55 (SEQ ID NO:448), HCN2 (SEQ ID NO:7377), NKAIN1 (SEQ ID NO:5039), DPP10 (SEQ ID NO:432), AJAP1 (SEQ ID NO:894), NPFFR1 (SEQ ID NO:4886), TRPC3 (SEQ ID NO:1434), TGFBI (SEQ ID NO:107), SLC6A7 (SEQ ID NO:7370), GABRA4 (SEQ ID NO:265), SLC13A5 (SEQ ID NO:1732), GRIN2C (SEQ ID NO:285), HCN1 (SEQ ID NO:4763), SLC26A8 (SEQ ID NO:5552), PPIC (SEQ ID NO:336), NETO1 (SEQ ID NO:7427), TNFRSF10B (SEQ ID NO:2910), CDH22 (SEQ ID NO:4803), SLC6A13 (SEQ ID NO:4264), DISP2 (SEQ ID NO:7320), SLC6A11 (SEQ ID NO:3890), CD93 (SEQ ID NO:3698), EPHA10 (SEQ ID NO:1160), PHLDB2 (SEQ ID NO:1490), OXTR (SEQ ID NO:7326), WNT7A (SEQ ID NO:3089), GYPC (SEQ ID NO:7432), KCNA4 (SEQ ID NO:2589), PCDHAC2 (SEQ ID NO:4498), HGFAC (SEQ ID NO:2405), DRD1 (SEQ ID NO:253), SHISA9 (SEQ ID NO:7418), SCN8A (SEQ ID NO:3879), ICAM1 (SEQ ID NO:7382), PIRT (SEQ ID NO:1201), A4GALT (SEQ ID NO:4291), MRGPRF (SEQ ID NO:1129), CD248 (SEQ ID NO:4649), CD58 (SEQ ID NO:7405), CD44 (SEQ ID NO:172), EPHA2 (SEQ ID NO:3042) or PROCR (SEQ ID NO:3480). In some instances, such antigen combinations may be useful in targeting melanoma.

Third Antigens

As noted above, in some cases, a genetically modified cytotoxic immune cell of the present disclosure, or a system of the present disclosure, can include an antigen-triggered polypeptide (or a nucleic acid comprising a nucleotide sequence encoding the same) that specifically binds a third target antigen present on the surface of a cancer cell. Examples of such third target antigens are depicted in FIG. 4 and Table 3. The third target antigen can be included except where contraindicated by a target antigen pair listed in FIG. 1 or FIG. 9-14 . In some cases, the third antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to one of the amino acid sequences depicted in SEQ ID NOs: 7119-7467.

Where a third antigen (e.g., an antigen listed in FIG. 4 or Table 3) is included in the target antigen combination, the third antigen will be associated with the same cancer cell type as the target antigen pair (e.g., from FIG. 1 or FIG. 9-14 ). FIG. 1 and FIG. 9-14 provide exemplary target cancer cell types associate with a given target antigen pair. FIG. 4 also provides exemplary target cancer cell types associate with a given target antigen.

In some instances, a particular antigen (e.g., an antigen of an antigen pair as described in FIG. 1 or FIG. 9-14 , a clinical antigen as described in FIG. 4 , etc.) may be employed in an antigen combination, including combinations having two antigens, combinations having three antigens, etc., for treating a cancer other than the exemplary cancer with which it is identified. For example, although FIG. 4 identifies “exemplary cancers” for each antigen, use of the antigen in antigen combinations will not be limited to the specifically identified cancer(s) and the particular antigen may be employed in antigen combinations for the treatment of other cancers besides those specifically listed as exemplary.

Examples of hematological malignancy antigens include, e.g., CD19 (as expressed in e.g., B-cells), CD20 (as expressed in e.g., B-cells), CD22 (as expressed in e.g., B-cells), CD30 (as expressed in e.g., B-cells), CD33 (as expressed in e.g., Myeloid cells), CD70 (as expressed in e.g., B-cell/T-cells), CD123 (as expressed in e.g., Myeloid cells), Kappa (as expressed in e.g., B-cells), Lewis Y (as expressed in e.g., Myeloid cells), NKG2D ligands (as expressed in e.g., Myeloid cells), ROR1 (as expressed in e.g., B-cells), SLAMF7/CS1 (as expressed in e.g., myeloma cells, natural killer cells, T cells, and most B-cell types), CD138 (as expressed in e.g., malignant plasma cells in multiple myelomas), CD56 (as expressed in e.g., myeloma cells, neural cells, natural killer cells, T cells, and trabecular osteoblasts) CD38 (as expressed in e.g., B-cell/T-cells) and CD160 (as expressed in e.g., NK cells/T-cells), and the like. Examples of solid tumor antigens include, e.g., B7H3 (as expressed in e.g., Sarcoma, glioma), CAIX (as expressed in e.g., Kidney), CD44 v6/v7 (as expressed in e.g., Cervical cancer), CD171 (as expressed in e.g., Neuroblastoma), CEA (as expressed in e.g., Colon), EGFRvIII (as expressed in e.g., Glioma), EGP2 (as expressed in e.g., Carcinomas), EGP40 (as expressed in e.g., Colon), EphA2 (as expressed in e.g., Glioma, lung), ErbB2(HER2) (as expressed in e.g., Breast, lung, prostate, glioma), ErbB receptor family (as expressed in e.g., Breast, lung, prostate, glioma), ErbB3/4 (as expressed in e.g., Breast, ovarian), HLA-A1/MAGE1 (as expressed in e.g., Melanoma), HLA-A2/NY-ESO-1 (as expressed in e.g., Sarcoma, melanoma), FR-a (as expressed in e.g., Ovarian), FAPt (as expressed in e.g., Cancer associated fibroblasts), FAR (as expressed in e.g., Rhabdomyosarcoma), GD2 (as expressed in e.g., Neuroblastoma, sarcoma, melanoma), GD3 (as expressed in e.g., Melanoma, lung cancer), HMW-MAA (as expressed in e.g., Melanoma), IL11Ra (as expressed in e.g., Osteosarcoma), IL13Ra2 (as expressed in e.g., Glioma), Lewis Y (as expressed in e.g., Breast/ovarian/pancreatic), Mesothelin (as expressed in e.g., Mesothelioma, breast, pancreas), Muc (as expressed in e.g., Ovarian, breast, prostate), NCAM (as expressed in e.g., Neuroblastoma, colorectal), NKG2D ligands (as expressed in e.g., Ovarian, sacoma), PSCA (as expressed in e.g., Prostate, pancreatic), PSMA (as expressed in e.g., Prostate), TAG72 (as expressed in e.g., Colon), VEGFR-2 (as expressed in e.g., Tumor vasculature), Axl (as expressed in e.g., Lung cancer), Met (as expressed in e.g., Lung cancer), α5β3 (as expressed in e.g., Tumor vasculature), α5β1 (as expressed in e.g., Tumor vasculature), TRAIL-R1/TRAIL-R2 (as expressed in e.g., Solid tumors (colon, lung, pancreas) and hematological malignancies), RANKL (as expressed in e.g., Prostate cancer and bone metastases), Tenacin (as expressed in e.g., Glioma, epithelial tumors (breast, prostate)), EpCAM (as expressed in e.g., Epithelial tumors (breast, colon, lung)), CEA (as expressed in e.g., Epithelial tumors (breast, colon, lung)), gpA33 (as expressed in e.g., Colorectal carcinoma), Mucins (as expressed in e.g., Epithelial tumors (breast, colon, lung, ovarian)), TAG-72 (as expressed in e.g., Epithelial tumors (breast, colon, lung)), EphA3 (as expressed in e.g., Lung, kidney, melanoma, glioma, hematological malignancies) and IGF1R (as expressed in e.g., Lung, breast, head and neck, prostate, thyroid, glioma). Examples of surface and intracellular antigens include, e.g., Her2 (gene symbol ERBB2), MAGE-A1 (gene symbol MAGEA1), MART-1 (gene symbol MLANA), NY-ESO (gene symbol CTAG1), WT1 (gene symbol WT1), MUC17 and MUC13. Examples of other antigens include, e.g., BCMA (gene symbol TNFRSF17), B7H6 (gene symbol NCR3LG1), CAIX (gene symbol CA9), CD123 (gene symbol IL3RA), CD138 (gene symbol SDC1), CD171 (gene symbol L1CAM), CD19 (gene symbol CD19), CD20 (gene symbol CD20), CD22 (gene symbol CD22), CD30 (gene symbol TNFRSF8), CD33 (gene symbol CD33), CD38 (gene symbol CD38), CD44, splice variants incl 7 and 8 (denoted vX in literature) (gene symbol CD44), CEA, CS1 (gene symbol SLAMF7), EGFRvIII (gene symbol EGFR, vIII deletion variant), EGP2, EGP40 (gene symbol EPCAM), Erb family member (gene symbol ERBB1, ERBB2, ERBB3, ERBB4), FAP (gene symbol FAP), fetal acetylcholine receptor (gene symbol AChR), Folate receptor alpha (gene symbol FOLR1), Folate receptor beta (gene symbol FOLR2), GD2, GD3, GPC3 (gene symbol GPC3), Her2/neu (gene symbol ERBB2), IL-13Ra2 (gene symbol IL13RA2), Kappa light chain (gene symbol IGK), Lewis-Y, Mesothelin (gene symbol MSLN), Mucin-1 (gene symbol MUC1), Mucin-16 (gene symbol MUC16), NKG2D ligands, prostate specific membrane antigen (PSMA) (gene symbol FOLH1), prostate stem cell antigen (PSCA) (gene symbol PSCA), receptor tyrosine kinase-like orphan receptor 1 (gene symbol ROR1), and Anaplastic Lymphoma Receptor Tyrosine Kinase (gene symbol ALK).

In some cases, useful third antigens may include an antigen selected from Table 3 above.

Useful three antigen combinations may or may not include a clinical antigen as an added “third antigen”. For example, in some instances, a useful three antigen combination may include a clinical antigen and two or more antigens that provide AND NOT functionality, including e.g., the combinations including a clinical antigen and two tissue specific AND NOT antigens (e.g., brain tissue and cardiac tissue) as described in more detail below. In some cases, useful three antigen combinations may not include a clinical antigen. Useful three antigen combinations may include various logic combinations including but not limited to e.g., antigen 1 AND antigen 2 AND antigen 3, antigen 1 AND antigen 2 AND NOT antigen 3, antigen 1 AND NOT antigen 2 AND NOT antigen 3, and the like. In some instances, the logic of a three antigen combination may be complex where “complex” logic, as used herein, refers to combinations having multiple positive prediction nodes in the associated tree or, e.g., where the logic contains one or more OR propositions. In some instances, useful three antigen combinations include those depicted in FIG. 15 .

In some cases, useful three antigen combinations include an antigen combination that includes a clinical antigen in combination with two or more tissue antigens (e.g., a first antigen expressed in normal tissue, e.g., normal brain tissue, and a second antigen expressed in normal tissue, e.g., normal cardiac tissue) in a double AND NOT gate (i.e., clinical antigen AND NOT normal tissue antigen 1 AND NOT normal tissue antigen 2).

For example, useful antigen combinations may include a MAGEA1 (SEQ ID NO:3191) clinical antigen AND NOT a cardiac tissue specific antigen (such as GJA3 (SEQ ID NO:4854), HCN4 (SEQ ID NO:3292) or BMP10 (SEQ ID NO:3945)) AND NOT a brain tissue antigen selected from: OPALIN (SEQ ID NO:833), TMEM235 (SEQ ID NO:1869), GABRA1 (SEQ ID NO:262), KCNJ9 (SEQ ID NO:3190), GRM3 (SEQ ID NO:290), SEZ6 (SEQ ID NO:1143), NTSR2 (SEQ ID NO:3753), KCNK4 (SEQ ID NO:5486), SLCO1A2 (SEQ ID NO:7290), SLC24A2 (SEQ ID NO:4632), MOG (SEQ ID NO:528), GABRG1 (SEQ ID NO:6354), GABRG2 (SEQ ID NO:272), CNTNAP4 (SEQ ID NO:5492), DSCAM (SEQ ID NO:2354), CACNG3 (SEQ ID NO:3510), CRB1 (SEQ ID NO:6891), CDH10 (SEQ ID NO:3552), HRH3 (SEQ ID NO:3657), GRIK1 (SEQ ID NO:280), SLC39A12 (SEQ ID NO:1826), GPR158 (SEQ ID NO:4716), CACNG2 (SEQ ID NO:3430), SYT3 (SEQ ID NO:7410), HTR5A (SEQ ID NO:4993), CACNG7 (SEQ ID NO:5264), GPR37L1 (SEQ ID NO:3118), LRRTM3 (SEQ ID NO:7362), GLRA2 (SEQ ID NO:1281), CHRNB2 (SEQ ID NO:228), KCNQ2 (SEQ ID NO:3067), JPH3 (SEQ ID NO:4690), GPR19 (SEQ ID NO:3446), ADCY8 (SEQ ID NO:1248), SPOCK3 (SEQ ID NO:845), SLC32A1 (SEQ ID NO:5606), OPCML (SEQ ID NO:594), GABRA3 (SEQ ID NO:264), GRM5 (SEQ ID NO:292), SCN1A (SEQ ID NO:2133), SLC5A11 (SEQ ID NO:5546), KCNC1 (SEQ ID NO:1249), SLC12A5 (SEQ ID NO:1506), GRM4 (SEQ ID NO:291), GRM1 (SEQ ID NO:288), GRIA4 (SEQ ID NO:279), MEGF11 (SEQ ID NO:5354), CACNA1B (SEQ ID NO:211), LYPD1 (SEQ ID NO:970), GRID2 (SEQ ID NO:2398), SCN2A (SEQ ID NO:842), NKAIN2 (SEQ ID NO:864), UNC5A (SEQ ID NO:5690), SLC4A10 (SEQ ID NO:7421), TMEFF2 (SEQ ID NO:4183), CSMD3 (SEQ ID NO:5532), PPAPDC1A (SEQ ID NO:715), HAPLN4 (SEQ ID NO:4968), GPR85 (SEQ ID NO:1920), ANTXR2 (SEQ ID NO:1876), CACNG4 (SEQ ID NO:3934), CSPG5 (SEQ ID NO:3516), KCNK10 (SEQ ID NO:4784), CHRNA4 (SEQ ID NO:225), CNTNAP2 (SEQ ID NO:3871), KCNJ10 (SEQ ID NO:2597), GABRB2 (SEQ ID NO:269), GRIN1 (SEQ ID NO:282), CRB2 (SEQ ID NO:6381), SHISA7 (SEQ ID NO:7406), NKAIN4 (SEQ ID NO:6122), HTR2C (SEQ ID NO:301), CACNG8 (SEQ ID NO:5263), NRG3 (SEQ ID NO:569), ABCG4 (SEQ ID NO:1668), CDH8 (SEQ ID NO:2476), GABRD (SEQ ID NO:271), KIRREL3 (SEQ ID NO:2022), GABRB1 (SEQ ID NO:268), KCNA2 (SEQ ID NO:3181), CDH20 (SEQ ID NO:5261), IGDCC3 (SEQ ID NO:3159), KCNJ6 (SEQ ID NO:2596), CNIH2 (SEQ ID NO:6658), KCNK12 (SEQ ID NO:7295), CDH18 (SEQ ID NO:2192), CSMD2 (SEQ ID NO:5530), SYT4 (SEQ ID NO:4722), OR2L13 (SEQ ID NO:6468), CDH9 (SEQ ID NO:4198), GABRA2 (SEQ ID NO:263), KCNF1 (SEQ ID NO:2592), MAG (SEQ ID NO:2634), CALN1 (SEQ ID NO:645), GRIN2B (SEQ ID NO:284), GRM7 (SEQ ID NO:294), VSTM2A (SEQ ID NO:7459), GPR61 (SEQ ID NO:5271), OMG (SEQ ID NO:2672), KCNA1 (SEQ ID NO:66), GPR83 (SEQ ID NO:4239), ATP8A2 (SEQ ID NO:4237), GABBR2 (SEQ ID NO:3288), GPR12 (SEQ ID NO:3255), TRPM3 (SEQ ID NO:519), SLC8A3 (SEQ ID NO:1424), KCND2 (SEQ ID NO:7288), GSG1L (SEQ ID NO:1236), SLC30A10 (SEQ ID NO:4487), ASTN1 (SEQ ID NO:3016), GPR179 (SEQ ID NO:7447), LRFN2 (SEQ ID NO:4711), CACNA1E (SEQ ID NO:214), CALY (SEQ ID NO:4119), SLC6A15 (SEQ ID NO:1932), KIAA0319 (SEQ ID NO:2227), SYT6 (SEQ ID NO:6957), PTPRR (SEQ ID NO:2724), KCTD8 (SEQ ID NO:7331), GPR22 (SEQ ID NO:3261), SLC4A8 (SEQ ID NO:818), LAMP5 (SEQ ID NO:3729), MEGF10 (SEQ ID NO:5355), FXYD7 (SEQ ID NO:4864), KCNK9 (SEQ ID NO:4257), SLC1A6 (SEQ ID NO:3206), MLC1 (SEQ ID NO:4042), OPRK1 (SEQ ID NO:325), ATP2B2 (SEQ ID NO:353), ACSL6 (SEQ ID NO:560), THBD (SEQ ID NO:108), PTPRT (SEQ ID NO:3622), PCDHGC4 (SEQ ID NO:2688), CLDN10 (SEQ ID NO:1975), KCNV1 (SEQ ID NO:3925), LPPR3 (SEQ ID NO:5096), SLCO1C1 (SEQ ID NO:1885), PCDH8 (SEQ ID NO:2690), ANO4 (SEQ ID NO:6566), LRRTM4 (SEQ ID NO:1505), PCDH15 (SEQ ID NO:1688), CCKBR (SEQ ID NO:6481), GABRA5 (SEQ ID NO:266), SLC6A12 (SEQ ID NO:1290), GRIN2A (SEQ ID NO:283), SLC1A2 (SEQ ID NO:2977), SLC43A1 (SEQ ID NO:2856), KCNC2 (SEQ ID NO:5836), ELFN2 (SEQ ID NO:5534), ATP7A (SEQ ID NO:10), GRIK4 (SEQ ID NO:3965), LRRC55 (SEQ ID NO:448), HCN2 (SEQ ID NO:7377), NKAIN1 (SEQ ID NO:5039), DPP10 (SEQ ID NO:432), AJAP1 (SEQ ID NO:894), NPFFR1 (SEQ ID NO:4886), TRPC3 (SEQ ID NO:1434), TGFBI (SEQ ID NO:107), SLC6A7 (SEQ ID NO:7370), GABRA4 (SEQ ID NO:265), SLC13A5 (SEQ ID NO:1732), GRIN2C (SEQ ID NO:285), HCN1 (SEQ ID NO:4763), SLC26A8 (SEQ ID NO:5552), PPIC (SEQ ID NO:336), NETO1 (SEQ ID NO:7427), TNFRSF10B (SEQ ID NO:2910), CDH22 (SEQ ID NO:4803), SLC6A13 (SEQ ID NO:4264), DISP2 (SEQ ID NO:7320), SLC6A11 (SEQ ID NO:3890), CD93 (SEQ ID NO:3698), EPHA10 (SEQ ID NO:1160), PHLDB2 (SEQ ID NO:1490), OXTR (SEQ ID NO:7326), WNT7A (SEQ ID NO:3089), GYPC (SEQ ID NO:7432), KCNA4 (SEQ ID NO:2589), PCDHAC2 (SEQ ID NO:4498), HGFAC (SEQ ID NO:2405), DRD1 (SEQ ID NO:253), SHISA9 (SEQ ID NO:7418), SCN8A (SEQ ID NO:3879), ICAM1 (SEQ ID NO:7382), PIRT (SEQ ID NO:1201), A4GALT (SEQ ID NO:4291), MRGPRF (SEQ ID NO:1129), CD248 (SEQ ID NO:4649), CD58 (SEQ ID NO:7405), CD44 (SEQ ID NO:172), EPHA2 (SEQ ID NO:3042) or PROCR (SEQ ID NO:3480). In some instances, such antigen combinations may be useful in targeting melanoma.

Useful antigen combinations may include a MAGEA3 (SEQ ID NO:7279) clinical antigen AND NOT a cardiac tissue specific antigen (such as GJA3 (SEQ ID NO:4854), HCN4 (SEQ ID NO:3292) or BMP10 (SEQ ID NO:3945)) AND NOT a brain tissue antigen selected from: OPALIN (SEQ ID NO:833), TMEM235 (SEQ ID NO:1869), GABRA1 (SEQ ID NO:262), KCNJ9 (SEQ ID NO:3190), GRM3 (SEQ ID NO:290), SEZ6 (SEQ ID NO:1143), NTSR2 (SEQ ID NO:3753), KCNK4 (SEQ ID NO:5486), SLCO1A2 (SEQ ID NO:7290), SLC24A2 (SEQ ID NO:4632), MOG (SEQ ID NO:528), GABRG1 (SEQ ID NO:6354), GABRG2 (SEQ ID NO:272), CNTNAP4 (SEQ ID NO:5492), DSCAM (SEQ ID NO:2354), CACNG3 (SEQ ID NO:3510), CRB1 (SEQ ID NO:6891), CDH10 (SEQ ID NO:3552), HRH3 (SEQ ID NO:3657), GRIK1 (SEQ ID NO:280), SLC39A12 (SEQ ID NO:1826), GPR158 (SEQ ID NO:4716), CACNG2 (SEQ ID NO:3430), SYT3 (SEQ ID NO:7410), HTR5A (SEQ ID NO:4993), CACNG7 (SEQ ID NO:5264), GPR37L1 (SEQ ID NO:3118), LRRTM3 (SEQ ID NO:7362), GLRA2 (SEQ ID NO:1281), CHRNB2 (SEQ ID NO:228), KCNQ2 (SEQ ID NO:3067), JPH3 (SEQ ID NO:4690), GPR19 (SEQ ID NO:3446), ADCY8 (SEQ ID NO:1248), SPOCK3 (SEQ ID NO:845), SLC32A1 (SEQ ID NO:5606), OPCML (SEQ ID NO:594), GABRA3 (SEQ ID NO:264), GRM5 (SEQ ID NO:292), SCN1A (SEQ ID NO:2133), SLC5A11 (SEQ ID NO:5546), KCNC1 (SEQ ID NO:1249), SLC12A5 (SEQ ID NO:1506), GRM4 (SEQ ID NO:291), GRM1 (SEQ ID NO:288), GRIA4 (SEQ ID NO:279), MEGF11 (SEQ ID NO:5354), CACNA1B (SEQ ID NO:211), LYPD1 (SEQ ID NO:970), GRID2 (SEQ ID NO:2398), SCN2A (SEQ ID NO:842), NKAIN2 (SEQ ID NO:864), UNC5A (SEQ ID NO:5690), SLC4A10 (SEQ ID NO:7421), TMEFF2 (SEQ ID NO:4183), CSMD3 (SEQ ID NO:5532), PPAPDC1A (SEQ ID NO:715), HAPLN4 (SEQ ID NO:4968), GPR85 (SEQ ID NO:1920), ANTXR2 (SEQ ID NO:1876), CACNG4 (SEQ ID NO:3934), CSPG5 (SEQ ID NO:3516), KCNK10 (SEQ ID NO:4784), CHRNA4 (SEQ ID NO:225), CNTNAP2 (SEQ ID NO:3871), KCNJ10 (SEQ ID NO:2597), GABRB2 (SEQ ID NO:269), GRIN1 (SEQ ID NO:282), CRB2 (SEQ ID NO:6381), SHISA7 (SEQ ID NO:7406), NKAIN4 (SEQ ID NO:6122), HTR2C (SEQ ID NO:301), CACNG8 (SEQ ID NO:5263), NRG3 (SEQ ID NO:569), ABCG4 (SEQ ID NO:1668), CDH8 (SEQ ID NO:2476), GABRD (SEQ ID NO:271), KIRREL3 (SEQ ID NO:2022), GABRB1 (SEQ ID NO:268), KCNA2 (SEQ ID NO:3181), CDH20 (SEQ ID NO:5261), IGDCC3 (SEQ ID NO:3159), KCNJ6 (SEQ ID NO:2596), CNIH2 (SEQ ID NO:6658), KCNK12 (SEQ ID NO:7295), CDH18 (SEQ ID NO:2192), CSMD2 (SEQ ID NO:5530), SYT4 (SEQ ID NO:4722), OR2L13 (SEQ ID NO:6468), CDH9 (SEQ ID NO:4198), GABRA2 (SEQ ID NO:263), KCNF1 (SEQ ID NO:2592), MAG (SEQ ID NO:2634), CALN1 (SEQ ID NO:645), GRIN2B (SEQ ID NO:284), GRM7 (SEQ ID NO:294), VSTM2A (SEQ ID NO:7459), GPR61 (SEQ ID NO:5271), OMG (SEQ ID NO:2672), KCNA1 (SEQ ID NO:66), GPR83 (SEQ ID NO:4239), ATP8A2 (SEQ ID NO:4237), GABBR2 (SEQ ID NO:3288), GPR12 (SEQ ID NO:3255), TRPM3 (SEQ ID NO:519), SLC8A3 (SEQ ID NO:1424), KCND2 (SEQ ID NO:7288), GSG1L (SEQ ID NO:1236), SLC30A10 (SEQ ID NO:4487), ASTN1 (SEQ ID NO:3016), GPR179 (SEQ ID NO:7447), LRFN2 (SEQ ID NO:4711), CACNA1E (SEQ ID NO:214), CALY (SEQ ID NO:4119), SLC6A15 (SEQ ID NO:1932), KIAA0319 (SEQ ID NO:2227), SYT6 (SEQ ID NO:6957), PTPRR (SEQ ID NO:2724), KCTD8 (SEQ ID NO:7331), GPR22 (SEQ ID NO:3261), SLC4A8 (SEQ ID NO:818), LAMP5 (SEQ ID NO:3729), MEGF10 (SEQ ID NO:5355), FXYD7 (SEQ ID NO:4864), KCNK9 (SEQ ID NO:4257), SLC1A6 (SEQ ID NO:3206), MLC1 (SEQ ID NO:4042), OPRK1 (SEQ ID NO:325), ATP2B2 (SEQ ID NO:353), ACSL6 (SEQ ID NO:560), THBD (SEQ ID NO:108), PTPRT (SEQ ID NO:3622), PCDHGC4 (SEQ ID NO:2688), CLDN10 (SEQ ID NO:1975), KCNV1 (SEQ ID NO:3925), LPPR3 (SEQ ID NO:5096), SLCO1C1 (SEQ ID NO:1885), PCDH8 (SEQ ID NO:2690), ANO4 (SEQ ID NO:6566), LRRTM4 (SEQ ID NO:1505), PCDH15 (SEQ ID NO:1688), CCKBR (SEQ ID NO:6481), GABRA5 (SEQ ID NO:266), SLC6A12 (SEQ ID NO:1290), GRIN2A (SEQ ID NO:283), SLC1A2 (SEQ ID NO:2977), SLC43A1 (SEQ ID NO:2856), KCNC2 (SEQ ID NO:5836), ELFN2 (SEQ ID NO:5534), ATP7A (SEQ ID NO:10), GRIK4 (SEQ ID NO:3965), LRRC55 (SEQ ID NO:448), HCN2 (SEQ ID NO:7377), NKAIN1 (SEQ ID NO:5039), DPP10 (SEQ ID NO:432), AJAP1 (SEQ ID NO:894), NPFFR1 (SEQ ID NO:4886), TRPC3 (SEQ ID NO:1434), TGFBI (SEQ ID NO:107), SLC6A7 (SEQ ID NO:7370), GABRA4 (SEQ ID NO:265), SLC13A5 (SEQ ID NO:1732), GRIN2C (SEQ ID NO:285), HCN1 (SEQ ID NO:4763), SLC26A8 (SEQ ID NO:5552), PPIC (SEQ ID NO:336), NETO1 (SEQ ID NO:7427), TNFRSF10B (SEQ ID NO:2910), CDH22 (SEQ ID NO:4803), SLC6A13 (SEQ ID NO:4264), DISP2 (SEQ ID NO:7320), SLC6A11 (SEQ ID NO:3890), CD93 (SEQ ID NO:3698), EPHA10 (SEQ ID NO:1160), PHLDB2 (SEQ ID NO:1490), OXTR (SEQ ID NO:7326), WNT7A (SEQ ID NO:3089), GYPC (SEQ ID NO:7432), KCNA4 (SEQ ID NO:2589), PCDHAC2 (SEQ ID NO:4498), HGFAC (SEQ ID NO:2405), DRD1 (SEQ ID NO:253), SHISA9 (SEQ ID NO:7418), SCN8A (SEQ ID NO:3879), ICAM1 (SEQ ID NO:7382), PIRT (SEQ ID NO:1201), A4GALT (SEQ ID NO:4291), MRGPRF (SEQ ID NO:1129), CD248 (SEQ ID NO:4649), CD58 (SEQ ID NO:7405), CD44 (SEQ ID NO:172), EPHA2 (SEQ ID NO:3042) or PROCR (SEQ ID NO:3480). In some instances, such antigen combinations may be useful in targeting melanoma.

Methods of Killing Target Cancer Cells

The present disclosure provides methods for killing a target cancer cell. The present disclosure provides a method of killing a target cancer cell in an individual. In some cases, a method of the present disclosure for killing a target cell in an individual comprises: a) introducing a system of the present disclosure into an immune cell (e.g., a CD8⁺ T cell; an NK cell) obtained from the individual, generating a modified immune cell; and b) administering the modified immune cell to the individual, where the modified immune cell kills the target cancer cell in the individual. In some cases, the modified cytotoxic T cell does not substantially kill non-target cells such as non-cancerous cells.

Example 1 provides an example of a method of killing a liposarcoma in an individual; and Example 2 provides an example of a method of killing a glioblastoma in an individual. This example is illustrative of how a CD8+ cell (or other immune cell) genetically modified to express first and second antigen-triggered polypeptides targeting any of the target antigen pairs depicted in FIG. 1 or FIG. 9-14 to target the corresponding cancer cells listed in FIG. 1 or FIG. 9-14 , can be used to kill a target cancer cell in an individual, and thereby to treat the cancer in the individual.

The present disclosure provides a method of killing a target cancer cell in an individual. In some cases, a method of the present disclosure for killing a target cell in an individual comprises administering a genetically modified cytotoxic immune cell (e.g., a genetically modified CD8⁺ T cell; a genetically modified NK cell) of the present disclosure to the individual, where the genetically modified immune cell kills the target cancer cell in the individual. In some cases, the modified cytotoxic T cell does not substantially kill non-target cells such as non-cancerous cells.

Where the target antigen pair targeted by a method of the present disclosure is an AND-NOT gate target antigen pair, a method of the present disclosure provides for killing of a target cancer cell, but not a non-cancerous cell. For example, in some cases, a method of the present disclosure provides for a ratio of killing of cancer cells to non-cancerous cells of at least 10:1, at least 15:1, at least 20:1 at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10³:1, at least 10⁴:1, or at least 10⁵:1.

Where the target antigen pair targeted by a method of the present disclosure is an AND gate target antigen pair, a method of the present disclosure provides for highly specific killing of a target cancer cell, and not a non-target (e.g., non-cancerous cell). For example, in some cases, a method of the present disclosure provides for a ratio of killing of cancer cells to non-cancerous cells of at least 10:1, at least 15:1, at least 20:1 at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 103:1, at least 10⁴:1, or at least 10⁵:1.

Methods Comprising Use of a System of the Present Disclosure

As noted above, in some cases, a method of the present disclosure for killing a target cell in an individual comprises: a) introducing a system of the present disclosure into an immune cell (e.g., a CD8+ T cell; an NK cell) obtained from the individual, generating a modified immune cell; and b) administering the modified immune cell to the individual, where the modified immune cell kills the target cancer cell in the individual. In some cases, the modified cytotoxic T cell does not substantially kill non-target cells such as non-cancerous cells.

T cells can be obtained from an individual (e.g., an individual having a cancer; an individual diagnosed as having a cancer; an individual being treated for a cancer with chemotherapy, radiation therapy, antibody therapy, surgery, etc.) using well-established methods. In some cases, a mixed population of cells is obtained from an individual; and CD8⁺ T cells and/or NK cells are isolated from the mixed population, such that a population of CD8⁺ T cells and/or NK cells is obtained that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 98% pure, i.e., the purified cell population includes less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 2%, of cells other than CD8⁺ T cells and or NK cells. A system of the present disclosure is then introduced into the purified CD8⁺ T cells and/or NK cells, to generate modified CD8⁺ T cells and/or modified NK cells that express the first antigen-triggered polypeptide and the second antigen-triggered polypeptide.

In some cases, as noted above, a system of the present disclosure comprises: a) a first antigen-triggered polypeptide that binds specifically to a first target antigen present on a target cancer cell; and b) a second antigen-triggered polypeptide that binds specifically to a second target antigen. In these instances, the polypeptides per se are introduced into an immune cell (e.g., CD8⁺ T cells and/or NK cells obtained from an individual). Methods of introducing polypeptides into a cell are known in the art; and any known method can be used. For example, in some cases, the first and the second antigen-triggered polypeptides comprise a protein transduction domain (PTD) at the N-terminus or the C-terminus of the polypeptides.

In some cases, as noted above, a system of the present disclosure comprises: a) a first nucleic acid comprising a nucleotide sequence encoding a first antigen-triggered polypeptide that binds specifically to a first target antigen present on a target cancer cell; and b) a second nucleic acid comprising a nucleotide sequence encoding a second antigen-triggered polypeptide that binds specifically to a second target antigen. In some cases, the first and the second antigen-triggered polypeptides are encoded by nucleotide sequences on separate nucleic acids. In other cases, the first and the second antigen-triggered polypeptides are encoded by nucleotide sequences present in the same nucleic acid. In some cases, the nucleic acid is a recombinant expression vector. In some cases, a system of the present disclosure comprises: a) a first recombinant expression vector comprising a nucleotide sequence encoding a first antigen-triggered polypeptide that binds specifically to a first target antigen present on a target cancer cell; and b) a second recombinant expression vector comprising a nucleotide sequence encoding a second antigen-triggered polypeptide that binds specifically to a second target antigen. In some cases, the nucleotide sequences are operably linked to a constitutive promoter. In some cases, the nucleotide sequences are operably linked to a regulatable promoter (e.g., an inducible promoter, a reversible promoter, etc.). In some cases, the nucleotide sequences are operably linked to an immune cell promoter, e.g., a T-cell specific promoter. In some cases, a system of the present disclosure comprises a recombinant expression vector comprising nucleotide sequences encoding: a) a first antigen-triggered polypeptide that binds specifically to a first target antigen present on a target cancer cell; and b) a second antigen-triggered polypeptide that binds specifically to a second target antigen. In some cases, the nucleotide sequences are operably linked to a constitutive promoter. In some cases, the nucleotide sequences are operably linked to a regulatable promoter (e.g., an inducible promoter, a reversible promoter, etc.). In some cases, the nucleotide sequences are operably linked to an immune cell promoter, e.g., a T-cell specific promoter. Suitable promoters include, but are not limited to; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; a metallothionein-I promoter; and various art-known promoters. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

In some instances, nucleic acids present in a system of the present disclosure include immune cell specific promoters that are expressed in one or more immune cell types, including but not limited to lymphocytes, hematopoietic stem cells and/or progeny thereof (i.e., immune cell progenitors), etc. Any convenient and appropriate promoter of an immune cell specific gene may find use in nucleic acids of the present disclosure. In some instances, an immune cell specific promoter of a nucleic acid present in a system of the present disclosure may be a T cell specific promoter. In some instances, an immune cell specific promoter of a nucleic acid present in a system of the present disclosure may be a light and/or heavy chain immunoglobulin gene promoter and may or may not include one or more related enhancer elements.

In some instances, an immune cell specific promoter of a nucleic acid present in a system of the present disclosure may be a promoter of a B29 gene promoter, a CD14 gene promoter, a CD43 gene promoter, a CD45 gene promoter, a CD68 gene promoter, a IFN-β gene promoter, a WASP gene promoter, a T-cell receptor β-chain gene promoter, a V9 γ (TRGV9) gene promoter, a V2 δ (TRDV2) gene promoter, and the like.

In some instances, an immune cell specific promoter present in a system of a nucleic acid of the present disclosure may be a viral promoter expressed in immune cells. As such, in some instances, viral promoters useful in nucleic acids present in a system of the present disclosure include viral promoters derived from immune cells viruses, including but not limited to, e.g., lentivirus promoters (e.g., human immunodeficiency virus (HIV), SIV, FIV, EIAV, or Visna promoters) including e.g., long terminal repeat (LTR) promoter, etc., Retroviridae promoters including, e.g., HTLV-I promoter, HTLV-II promoter, etc., and the like.

In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, a CD4 gene promoter can be used; see, e.g., Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739; and Marodon et al. (2003) Blood 101:3416. As another example, a CD8 gene promoter can be used. NK cell-specific expression can be achieved by use of an Ncr1 (p46) promoter; see, e.g., Eckelhart et al. (2011) Blood 117:1565.

Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable recombinant expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

A method of the present disclosure for killing a target cell in an individual comprising: a) introducing a system of the present disclosure into an immune cell (e.g., a CD8⁺ T cell; an NK cell) obtained from the individual, generating a modified immune cell; and b) administering the modified immune cell to the individual, where the modified immune cell kills the target cancer cell in the individual, involves administering an effective amount of the modified immune cells to the individual.

In some cases, an effective amount (e.g., an effective number) of modified immune cells is an amount that, when administered in one or more doses to an individual having a cancer, decreases the number of cancer cells in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least 98%, compared to the number of cancer cells in the individual before said administration.

In some cases, from about 10² to about 10⁹ modified immune cells are administered to an individual in a single dose. In some cases, a single dose of modified immune cells disclosure contains from 10² to about 10⁴, from about 10⁴ to about 10⁵, from about 10⁵ to about 10⁶, from about 10⁶ to about 10⁷, from about 10⁷ to about 10⁸, or from about 10⁸ to about 10⁹ modified immune cells. In some cases, a single dose of modified immune cells is administered. Multiple doses can also be administered, as needed and/or as determined by a medical professional. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, doses can be administered. If multiple doses are administered, the multiple doses can be administered at various frequencies, including, e.g., once per week, twice per month, once per month, once every 2 months, once every 3 months, once every 4 months, once every 6 months, or once per year.

In some cases, the target cancer cell is a liposarcoma, a glioblastoma, a breast cancer cell, a renal cancer cell, a pancreatic cancer cell, a melanoma, an anaplastic lymphoma, a leiomyosarcoma, an astrocytoma, an ovarian cancer cell, a neuroblastoma, a mantle cell lymphoma, a sarcoma, a non-small cell lung cancer cell, an acute myeloid leukemia (AML) cell, a stomach cancer cell, a B-cell cancer cell, a lung cancer cell, or an oligodendroglioma.

Methods Comprising Use of a Genetically Modified Cytotoxic T Cell of the Present Disclosure

As noted above, in some cases, a method of the present disclosure for killing a target cell in an individual comprises administering a genetically modified cytotoxic immune cell (e.g., a genetically modified CD8⁺ T cell; a genetically modified NK cell) of the present disclosure to the individual, where the genetically modified immune cell kills the target cancer cell in the individual. In some cases, the modified cytotoxic T cell does not substantially kill non-target cells such as non-cancerous cells.

T cells can be obtained from an individual (e.g., an individual having a cancer; an individual diagnosed as having a cancer; an individual being treated for a cancer with chemotherapy, radiation therapy, antibody therapy, surgery, etc.) using well-established methods. In some cases, a mixed population of cells is obtained from an individual; and CD8⁺ T cells and/or NK cells are isolated from the mixed population, such that a population of CD8⁺ T cells and/or NK cells is obtained that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 98% pure, i.e., the purified cell population includes less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 2%, of cells other than CD8⁺ T cells and or NK cells. The purified CD8⁺ T cells and/or NK cells are then genetically modified to express the first antigen-triggered polypeptide and the second antigen-triggered polypeptide.

A method of the present disclosure for killing a target cell in an individual comprising administering a genetically modified cytotoxic immune cell (e.g., a genetically modified CD8⁺ T cell; a genetically modified NK cell) of the present disclosure to the individual involves administering an effective amount of a genetically modified cytotoxic immune cell of the present disclosure to the individual.

In some cases, an effective amount (e.g., an effective number) of genetically modified cytotoxic immune cells of the present disclosure is an amount that, when administered in one or more doses to an individual having a cancer, decreases the number of cancer cells in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least 98%, compared to the number of cancer cells in the individual before said administration.

In some cases, from about 10² to about 10⁹ genetically modified cytotoxic immune cells of the present disclosure are administered to an individual in a single dose. In some cases, a single dose of genetically modified cytotoxic immune cells of the present disclosure contains from 10² to about 10⁴, from about 10⁴ to about 10⁵, from about 10⁵ to about 10⁶, from about 10⁶ to about 10⁷, from about 10⁷ to about 10⁸, or from about 10⁸ to about 10⁹ genetically modified cytotoxic immune cells of the present disclosure. In some cases, a single dose of genetically modified cytotoxic immune cells of the present disclosure is administered. Multiple doses can also be administered, as needed and/or as determined by a medical professional. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, doses can be administered. If multiple doses are administered, the multiple doses can be administered at various frequencies, including, e.g., once per week, twice per month, once per month, once every 2 months, once every 3 months, once every 4 months, once every 6 months, or once per year.

In some cases, the target cancer cell is a liposarcoma, a glioblastoma, a breast cancer cell, a renal cancer cell, a pancreatic cancer cell, a melanoma, an anaplastic lymphoma, a leiomyosarcoma, an astrocytoma, an ovarian cancer cell, a neuroblastoma, a mantle cell lymphoma, a sarcoma, a non-small cell lung cancer cell, an acute myeloid leukemia (AML) cell, a stomach cancer cell, a B-cell cancer cell, a lung cancer cell, or an oligodendroglioma.

Individuals Suitable for Treatment

Individuals suitable for treatment using a method of the present disclosure include an individual having a cancer; an individual diagnosed as having a cancer; an individual being treated for a cancer with chemotherapy, radiation therapy, antibody therapy, surgery, etc.); an individual who has been treated for a cancer (e.g., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.), and who has failed to respond to the treatment; an individual who has been treated for a cancer (e.g., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.), and who initially responded to the treatment but who subsequently relapsed, i.e., the cancer recurred.

Cancers that can be treated with a method of the present disclosure include liposarcoma, glioblastoma, breast cancer, renal cancer, pancreatic cancer, melanoma, anaplastic lymphoma, leiomyosarcoma, astrocytoma, ovarian cancer, neuroblastoma, mantle cell lymphoma, sarcoma, non-small cell lung cancer, acute myeloid leukemia (AML), stomach cancer, B-cell cancer, lung cancer, and oligodendroglioma.

In some cases, an individual to which a treatment of the present disclosure is administered is an individual expressing one or more antigens relevant to the subject treatment, including e.g., one or more target (i.e., cancer) antigens and/or one or more non-target (i.e., non-cancer or normal) antigens. Antigen expression may be determined by any convenient means. For example, in some instances, a subject may be evaluated for expression (or lack thereof) of one or more antigens relevant to the subject treatment, including one or more or all of the antigens of a particular antigen combination utilized in the treatment. Such evaluations (i.e., antigen expression testing) may be performed at any convenient time before, during or after a particular treatment regimen and using any convenient sample obtained from a subject (e.g., a tissue sample, a biopsy sample, etc.). Evaluations of antigen expression may be employed predictively (e.g., to predict the efficacy of an antigen combination based therapy), concurrently (e.g., to confirm the expression of antigens of an antigen combination during therapy), retrospectively (e.g., to analyze the expression of antigens of an antigen combination after therapy, e.g., to correlate expression of treatment outcomes, e.g., as part of a clinical trial utilizing an antigen combination described herein), or the like.

Computational Methods

The instant disclosure includes computational methods of identifying antigen combinations for targeting cancer cells of various types. The computational methods will generally be based on the measured expression of a plurality of cell surface antigens and/or genes encoding cell surface antigens for a first specific population of cells relative to a second specific population of cells. For example, in some instances, the cell surface antigen expression within a population of cancer cells is measured relative to the cell surface antigen expression within a population of corresponding cells, where corresponding cells will generally be non-cancerous cells of the same type or derived from the same tissue as the cancer cells. In certain cases, gene or protein expression is compared between two tissues of the same type where one tissue contains a cancer and the other tissue is healthy or is otherwise suspected to not contain the cancer. In some cases, cell surface antigen expression within a population of cancer cells or a cancerous tissue is measured relative to the cell surface antigen expression within a population of corresponding cells or to the cell surface antigen expression within a corresponding tissue, where corresponding cells or tissue will generally be non-cancerous cells or tissue of the same type or derived from the same tissue as the cancer cells; in some cases, the non-cancerous cells (or normal tissue) are cells or tissue for which cell surface antigen expression data are publicly available. The antigen expression level(s) for a particular population of cells or tissue may be referred to as an expression dataset (e.g., a cancer tissue dataset, a normal tissue data set, a cancer cell dataset, a normal cell dataset, etc.).

Any convenient method may find use in comparing antigen expression of the cells and/or tissues, including methods for evaluating mRNA expression and methods for evaluating protein expression, including but not limited to e.g., RNA microarray methods, quantitative RNA sequencing (RNAseq) methods, quantitative polymerase chain reaction (qPCR) methods, protein microarray methods, quantitative mass-spec method, proteomics methods, and the like. In certain cases, existing data sets including but not limited to e.g., RNA microarray data sets, quantitative RNA sequencing (RNAseq) data sets, qPCR data sets, protein microarray data sets, quantitative mass-spec data sets, proteomics methods, etc., may be used in the computational methods as described herein. In some cases, a combination of two or three of these methods is used for comparing antigen expression of cells/tissues. In some instances, the data set may be a curated, including a human curated dataset or computationally annotated dataset. In some cases, single cell measurements, or micro dissected data, are used. In some cases, multiple (2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) datasets are used. In some cases, e.g., where multiple data sets are used, each sample within each dataset is curated.

In some embodiments, an expression dataset comparing a cancerous biological sample vs. a corresponding normal biological sample may be utilized in the subject computational methods. A “biological sample” encompasses a variety of sample types obtained from an individual. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, microdissection, or enrichment for certain components, such as polynucleotides or polypeptides. A “biological sample” can be a single cell, or can be derived from a single cell. A “biological sample” can be a plurality of cells, or can be derived from a plurality of cells. The term “biological sample” encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples. The term “biological sample” includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, bronchoalveolar lavage fluid, blood fractions such as plasma and serum, and the like, provided such samples contain the subject cells for which expression is to be measured. The term “biological sample” also includes solid tissue samples, tissue culture samples, and cellular samples.

Following procurement of expression datasets, whether measured de novo or obtained from a previously collected dataset or a combination thereof, the datasets will generally be normalized. In some cases, normalization is performed by computing the relative levels of expression to a reference gene or reference gene set. In some cases, normalization is performed by comparing all measurements of a dataset as a whole or globally, e.g., by computationally modeling the dataset. Useful normalization methods will vary and may include but are not limited to e.g., Single-channel array normalization (SCAN), Universal exPression Codes (UPC) normalization, and the like, as described in e.g., Piccolo et al. (2012) Genomics, 100(6), pp. 337-344; Piccolo et al. (2013) PNAS, 110(44), pp. 17778-17783; the disclosures of which are incorporated herein by reference in their entirety. Normalization may allow not only for comparison of data points within a dataset but also between two or more datasets.

An expression dataset may be used to produce parameters for a target antigen and/or target antigen pairs. Useful target antigen parameters include, but are not limited to, the relative expression levels of a particular antigen and/or the relative expression levels of a pair of antigens compared to other antigen pairs derived from the data set. Useful target antigen parameters include, but are not limited to, relative expression levels of a particular antigen on a cancer cell or cancerous tissue compared to the expression levels of the antigen on a corresponding non-cancerous cell of the same cell type or corresponding non-cancerous tissues of the same tissue type and/or relative expression levels of a pair of antigens on a cancer cell or cancerous tissue compared to their levels on a corresponding non-cancerous cell of the same cell type or corresponding non-cancerous tissues of the same tissue type.

In some cases, a portion of the data (e.g., a portion of a larger dataset) is selected for training of a processor used in generating an algorithm for identifying antigen pairs. Such a selected portion of the data (e.g., a selected portion of a larger dataset) may be referred to as a “training data set” and may include data of various types including but not limited to e.g., any of those described herein. In using a training dataset, individual antigen expression levels may be compared pairwise to identify significant differences between the datasets and/or pairs of antigen expression levels may be compared pairwise to identify significant differences between the antigen pairs of the datasets. Such comparisons may be performed iteratively using the training set and/or may be expanded to include the entire dataset or datasets. From a training set of expression data, antigens and/or antigen pairs that discriminate between cell or tissue populations (e.g., cancer vs. non-cancer) may be selected and used in generating an algorithm for selecting and/or ranking antigens and/or antigen pairs of one or more larger datasets.

In some instances, the pairing algorithm may be constrained to select one or more antigens for each pairing that meets a specified criteria including e.g., where one of the antigens is a clinical antigen (i.e., has been evaluated clinically (i.e., in an immunotherapy clinical trial) and found to be a marker for one or more cancers), where two or more of the antigens are clinical antigens, where one or more of the antigens is not a clinical antigen, where two or more of the antigens are not clinical antigens, where one of the antigens is a clinical antigen and one of the antigens is not a clinical antigen, etc. In some instances, the pairing algorithm may be constrained to select only those antigens that contain a transmembrane domain, including or excluding putative transmembrane domains. In some cases, a clinical antigen that has been the target of immunotherapy for a first cancer cell type can be identified, using a computational method of the present disclosure, as a target antigen for a second cancer cell type (i.e., a cancer cell type that is different from the first cancer cell type).

An algorithm for identifying antigen combinations (e.g., antigen pairs) useful in antigen logic gates of the subject disclosure may include a decision-tree classifier, where the training dataset may be used to develop an algorithm that identifies a classification model that best fits the antigen parameters to the class labels (i.e., the populations) of the training dataset. Decision tree classifiers are organized as a series of test questions to separate antigens based on their different characteristics or expression parameters. The decision tree may iteratively test antigen pairs for the ability to differentiate a cancer cell from a non-cancer cell. A non-limiting examples of such decision trees are provided in FIG. 7 , which schematically depicts a decision tree for identifying an AND-AND gate antigen pair, and FIG. 8 , which schematically depicts a decision tree for identifying an AND-NOT gate antigen pair.

Various calculated performance measures or statistical values, and/or combinations thereof, may find use in an algorithm to identify useful antigen combinations. For example, in some instances, an algorithm, as described herein, may include a measure of the fraction of retrieved antigen combinations that are relevant (e.g., a precision statistic or a positive predictive value statistic) and/or a measure of the fraction of relevant antigen combinations that are retrieved (e.g., a recall statistic or a sensitivity statistic). In some instances, an algorithm, as described herein, may include a combined statistic of precision and recall including but not limited to e.g., the harmonic mean of precision and recall or, e.g., a F-score such as e.g., an F1 statistic, where the F1 statistic is calculated as ((the product of the precision and recall statistics) divided by (the sum or the precision and recall statistics)) multiplied by two. Precision, recall and F-score (e.g., F1 statistic), as well as various other statistical measures useful in an antigen combination determining algorithm that may be employed, include but are not limited to e.g., those described in Pizzuti et al. Evolutionary Computation, Machine Learning and Data Mining in Bioinformatics: 9th European Conference, EvoBIO (2011), Torino, Italy, Apr. 27-29, 2011; Lee J K, Statistical Bioinformatics: For Biomedical and Life Science Researchers (2011), John Wiley & Sons; Mirkin B, Core Concepts in Data Analysis: Summarization, Correlation and Visualization (2011) Springer Science & Business Media; Kohane et al. Microarrays for an Integrative Genomics (2005) MIT Press; and the like.

The precision statistic, in this context, may be used to indicate the proportion of cancer samples of those samples from the test data that are predicted to trigger a cytotoxic response. Normal samples with expression patterns that correspond to the targeted logic may count as false positives and may impact the precision score. As normal samples come not only from multiple individuals, but from a wide variety of distinct body tissues, precision can be used to describe the level of risk of unwanted off-tumor reactivity associated with a particular targeting configuration.

The recall statistic, in this context, may be used to indicate the proportion of cancer samples in the test data with expression patterns that conform to the desired targeting logic. Cancer samples in the test data with expression patterns that do not match the targeting logic may count as false negatives and impact the recall score. As tumor samples come from different tumors or patients, they reflect heterogeneity both of tumors and individuals. Therefore recall can be used to describe the efficacy or generalizability of a given targeting configuration.

An F-score (e.g., an F1 statistic) represents a combination of precision and recall, that, in this context, captures the tradeoff between safety and efficacy, and allows for a summary of the performance of different configurations along a single axis. Various constants for the F-score may be employed that will combine precision and recall in such a way as to emphasize one or the other, depending on the particular context.

Any convenient calculated statistic may be used independently or in combination as a cutoff value for computationally identifying useful antigen combinations as described herein. For example, in some instances, a precision statistic may be used as a precision cutoff in an algorithm for identifying a useful antigen combination. Useful precision cutoff values will vary depending on the particular computational method employed and may range from less than 0.7 to 1.0 including but not limited to e.g., 0.7 to 1.0, 0.75 to 1.0, 0.76 to 1, 0.77 to 1, 0.78 to 1, 0.79 to 1, 0.8 to 1, 0.81 to 1, 0.82 to 1, 0.83 to 1, 0.84 to 1, 0.85 to 1, 0.86 to 1, 0.87 to 1, 0.88 to 1, 0.89 to 1, 0.9 to 1, 0.91 to 1, 0.92 to 1, 0.93 to 1, 0.94 to 1, 0.95 to 1, 0.96 to 1, 0.97 to 1, 0.98 to 1, etc. In some cases, the precision cutoff values can range from 0.5 to 0.7 or 0.5 to 1.0; e.g., from 0.55 to 0.7, from 0.60 to 0.7, from 0.5 to 1.0, from 0.6 to 1.0, or from 0.65 to 0.7.

In some instances, a recall statistic may be used as a recall cutoff in an algorithm for identifying a useful antigen combination. Useful recall cutoff values will vary depending on the particular computational method employed and may range from less than 0.7 to 1.0 including but not limited to e.g., 0.7 to 1.0, 0.75 to 1.0, 0.76 to 1, 0.77 to 1, 0.78 to 1, 0.79 to 1, 0.8 to 1, 0.81 to 1, 0.82 to 1, 0.83 to 1, 0.84 to 1, 0.85 to 1, 0.86 to 1, 0.87 to 1, 0.88 to 1, 0.89 to 1, 0.9 to 1, 0.91 to 1, 0.92 to 1, 0.93 to 1, 0.94 to 1, 0.95 to 1, 0.96 to 1, 0.97 to 1, 0.98 to 1, etc.

In some instances, an F-score (such as an F1 statistic for example) may be used as a F1 cutoff in an algorithm for identifying a useful antigen combination. Useful F1 cutoff values will vary depending on the particular computational method employed and may range from less than 0.8 to 1.0 including but not limited to e.g., 0.8 to 1.0, 0.81 to 1, 0.82 to 1, 0.83 to 1, 0.84 to 1, 0.85 to 1, 0.86 to 1, 0.87 to 1, 0.88 to 1, 0.89 to 1, 0.9 to 1, 0.91 to 1, 0.92 to 1, 0.93 to 1, 0.94 to 1, 0.95 to 1, 0.96 to 1, 0.97 to 1, 0.98 to 1, 0.99 to 1, etc. In some cases, the F1 cutoff values can range from 0.5 to 1.0; e.g., from 0.55 to 0.7, from 0.60 to 0.7, from 0.5 to 1.0, from 0.6 to 1.0, from 0.65 to 0.8, or from 0.7 to 1.0.

In some instances, two or more statistics, including two or more different statistics may be used in combination including but not limited to e.g., 2 or more different statistics, 3 or more different statistics, 4 or more different statistics, 5 or more different statistics, 6 or more different statistics, 7 or more different statistics, 8 or more different statistics, 9 or more different statistics, 10 or more different statistics, etc.

Examples of Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered as below are provided. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

1. An in vitro or ex vivo genetically modified cytotoxic immune cell, wherein the cytotoxic immune cell is genetically modified to produce two different polypeptides that recognize two different cell surface antigens, wherein at least one of the two different cell surface antigens is present on the surface of a target cancer cell.

2. The genetically modified cytotoxic immune cell of aspect 1, wherein the two different polypeptides comprise: a) a first antigen-triggered polypeptide that binds specifically to a first target cell surface antigen present on a target cancer cell; and b) a second antigen-triggered polypeptide that binds specifically to a second target cell surface antigen.

3. The genetically modified cytotoxic immune cell of aspect 1 or aspect 2, wherein the cytotoxic immune cell is a cytotoxic T cell or a natural killer cell.

4. The genetically modified cytotoxic immune cell of any one of aspect 1-3, wherein the immune cell is activated to kill a target cancer cell only when the target cancer cell expresses both of the two different cell surface antigens on its cell surface.

5. The genetically modified cytotoxic immune cell of aspect 2 or aspect 3, wherein the cytotoxic immune cell: a) is activated to kill a target cancer cell that expresses the first target cell surface antigen, but not the second target cell surface antigen, on its cell surface; and b) is inhibited from killing a non-cancerous cell if the non-cancerous cell expresses both the first target cell surface antigen and the second target cell surface antigen on its cell surface.

6. The genetically modified cytotoxic immune cell any one of aspects 2-5, wherein the first antigen-triggered polypeptide is a synNotch receptor and wherein the second antigen-triggered polypeptide is a chimeric antigen receptor (CAR). In some cases, the CAR is an ON-switch CAR.

7. The genetically modified cytotoxic immune cell any one of aspects 2-5, wherein the first antigen-triggered polypeptide is a synNotch receptor and wherein the second antigen-triggered polypeptide is a T cell receptor (TCR).

8. The genetically modified cytotoxic immune cell any one of aspects 2-5, wherein the first antigen-triggered polypeptide is a CAR, and wherein the second antigen-triggered polypeptide is an iCAR.

9. The genetically modified cytotoxic immune cell any one of aspects 2-5, wherein the first antigen-triggered polypeptide is a CAR, and wherein the second antigen-triggered polypeptide is a synNotch receptor.

10. The genetically modified cytotoxic immune cell any one of aspects 2-5, wherein the first antigen-triggered polypeptide is a synNotch receptor and wherein activation of the synNotch receptor induces expression of the second antigen-triggered polypeptide.

11. The genetically modified cytotoxic immune cell any one of aspects 2-5, wherein the first antigen-triggered polypeptide is a synNotch receptor and wherein activation of the synNotch receptor induces expression of an immune inhibitory polypeptide.

12. The genetically modified cytotoxic immune cell any one of aspects 1-11, wherein the target cancer cell is a liposarcoma, a glioblastoma, a breast cancer cell, a renal cancer cell, a pancreatic cancer cell, a melanoma, an anaplastic lymphoma, a leiomyosarcoma, an astrocytoma, an ovarian cancer cell, a neuroblastoma, a mantle cell lymphoma, a sarcoma, a non-small cell lung cancer cell, an AML cell, a stomach cancer cell, a B-cell cancer cell, a lung cancer cell, or an oligodendroglioma.

13. The genetically modified cytotoxic immune cell any one of aspects 1-12, wherein the two different cell surface antigens are a target antigen pair selected from a target antigen pair depicted in FIG. 1 or FIG. 9-14 .

14. The genetically modified cytotoxic immune cell of any one of aspects 1-13, wherein the genetically modified to produce a third antigen-triggered polypeptide that recognizes an additional cell surface antigen present on the target cancer cell.

15. The genetically modified cytotoxic immune cell of aspect 14, wherein the additional cell surface antigen is selected from an antigen depicted in FIG. 4 or Table 3.

16. The genetically modified cytotoxic immune cell of aspect 14, wherein the additional cell surface antigen is selected from: a) an α-folate receptor; b) carbonic anhydrase IX (CAIX); c) CD19; d) CD20; e) CD22; f) CD33; g) CD44v7/8; h) carcinoembryonic antigen; i) epithelial glycoprotein-2 (EGP-2); j) epithelial glycoprotein-40 (EGP-40); k) erb-B2; l) folate binding protein (FBP); m) fetal acetylcholine receptor; n) GD2 ganglioside; o) GD3 ganglioside; p) Her2/neu; q) IL-13R-a2; r) kinase insert domain receptor (KDR); s) immunoglobulin kappa light chain; t) LeY; u) melanoma antigen E-A1 (MAGE-A1); v) mesothelin; w) epithelial mucin MUC1; x) prostate stem cell antigen (PSCA); y) prostate-specific membrane antigen (PSMA); z) tumor-associated glycoprotein-72 (TAG-72); and aa) vascular endothelial growth factor receptor-2 (VEGF-R2).

17. A method of killing a target cancer cell in an individual, the method comprising administering to the individual an effective number of the genetically modified cytotoxic immune cell of any one of aspects 1-16, wherein said genetically modified cytotoxic immune cell kills the target cancer cell in the individual.

18. The method of aspect 17, wherein the target cancer cell is a liposarcoma, a glioblastoma, a breast cancer cell, a renal cancer cell, a pancreatic cancer cell, a melanoma, an anaplastic lymphoma, a leiomyosarcoma, an astrocytoma, an ovarian cancer cell, a neuroblastoma, a mantle cell lymphoma, a sarcoma, a non-small cell lung cancer cell, an AML cell, a stomach cancer cell, a B-cell cancer cell, a lung cancer cell, or an oligodendroglioma.

19. A system for killing a target cancer cell, the system comprising: a) a first antigen-triggered polypeptide that binds specifically to a first target antigen present on the target cancer cell, or a first nucleic acid comprising a nucleotide sequence encoding the first antigen-triggered polypeptide; and b) a second antigen-triggered polypeptide that binds specifically to a second target antigen, or a second nucleic acid comprising a nucleotide sequence encoding the second antigen-triggered polypeptide. In some cases, the system comprises: a) a first nucleic acid comprising a nucleotide sequence encoding first antigen-triggered polypeptide that binds specifically to a first target antigen present on the target cancer cell; and b) a second nucleic acid comprising a nucleotide sequence encoding a second antigen-triggered polypeptide that binds specifically to a second target antigen. In some cases, the first and the second nucleic acids are recombinant expression vectors. In some cases, the first and the second nucleic acids are present in separate recombinant expression vectors. In some cases, the first and the second nucleic acids are present in a single recombinant expression vectors. In some cases, the nucleotide sequences are operably linked to promoters. In some cases, the promoters are T-cell-specific promoters. In some cases, the promoters are constitutive promoters. In some cases, the promoters are regulatable promoters.

20. The system of aspect 19, wherein the first antigen-triggered polypeptide is a synNotch polypeptide, and the second antigen-triggered polypeptide is a chimeric antigen receptor (CAR).

21. The system of aspect 19, wherein the first antigen-triggered polypeptide is a synNotch polypeptide, and the second antigen-triggered polypeptide is an inhibitory CAR (iCAR).

22. The system of aspect 19, wherein the first antigen-triggered polypeptide is a synNotch polypeptide, and the second antigen-triggered polypeptide is a split CAR.

23. The system of aspect 19, wherein the first target antigen and the second target antigen are both present on the surface of a target cancer cell.

24. The system of aspect 19, wherein: a) the first target antigen and the second target antigen are both present on the surface of a non-cancerous cell; and b) wherein the first target antigen, but not the second target antigen, is present on the surface of a target cancer cell.

25. The system of any one of aspects 19-24, wherein the first target antigen and the second target antigen are selected from a target antigen pair depicted in FIG. 1 or FIG. 9-14 .

26. The system of any one of aspects 19-24, wherein the target cancer cell is a liposarcoma, a glioblastoma, a breast cancer cell, a renal cancer cell, a pancreatic cancer cell, a melanoma, an anaplastic lymphoma, a leiomyosarcoma, an astrocytoma, an ovarian cancer cell, a neuroblastoma, a mantle cell lymphoma, a sarcoma, a non-small cell lung cancer cell, an AML cell, a stomach cancer cell, a B-cell cancer cell, a lung cancer cell, or an oligodendroglioma.

27. The system of aspect 19, wherein the second target antigen is present on the target cancer cell.

28. The system of aspect 19, wherein the second target antigen is not present on the target cancer cell.

29. The system of aspect 19, wherein the first antigen-triggered polypeptide induces production of an antibody upon binding to the first target antigen.

30. The system of aspect 19, wherein the first antigen-triggered polypeptide induces production of a cytokine upon binding to the first target antigen.

31. A method of killing a target cancer cell in an individual, the method comprising: a) introducing the system of any one of aspects 19-30 into a cytotoxic T cell in vitro or ex vivo, generating a modified cytotoxic T cell; and b) administering the modified cytotoxic T cell to the individual.

32. The method of aspect 31, wherein the target cancer cell is a liposarcoma, a glioblastoma, a breast cancer cell, a renal cancer cell, a pancreatic cancer cell, a melanoma, an anaplastic lymphoma, a leiomyosarcoma, an astrocytoma, an ovarian cancer cell, a neuroblastoma, a mantle cell lymphoma, a sarcoma, a non-small cell lung cancer cell, an AML cell, a stomach cancer cell, a B-cell cancer cell, a lung cancer cell, or an oligodendroglioma.

33. The method of aspect 31 or 32, wherein the modified cytotoxic T cell is activated to kill the target cancer cell only when the target cancer cell expresses both the first target antigen and the second target antigen on its cell surface.

34. The method of aspect 31 or 32, wherein the modified cytotoxic T cell: a) is activated to kill a target cancer cell that expresses the first target cell surface antigen, but not the second target cell surface antigen, on its cell surface; and b) is inhibited from killing a non-cancerous cell if the non-cancerous cell expresses both the first target cell surface antigen and the second target cell surface antigen on its cell surface.

35. A method of identifying a combination of target antigens for targeting a cancer cell, the method comprising: a) generating a training set of target antigen parameters by determining, from a training set of expression data, pairs of antigens that discriminate between cancer cells and non-cancerous cells, thereby generating an algorithm; b) applying the algorithm to a test set of expression data, to generate a combination of target antigens.

36. The method of aspect 35, wherein step (a) comprises selecting pairs of target antigens that comprise: i) a first target antigen that is expressed at a high level on cancer cells of a selected cancer cell type; and ii) a second target antigen that is expressed at a high level on cancer cells of the selected cancer cell type; or i) a first target antigen that is expressed at a high level on cancer cells of a selected cancer cell type and on non-cancerous cells of the same cell type; and ii) a second target antigen that is expressed at a high level on non-cancerous cells of the same cell type but not on cancer cells of the same cell type.

37. The method of aspects 35 or 36, wherein the algorithm comprises one or more of an F1 cutoff, a precision cutoff, a recall cutoff, or combination thereof.

38. The method of any of aspects 35 to 37, wherein step (a) comprises training a computer software with the training set, and wherein step (b) comprises applying the algorithm using the trained computer software.

39. The method of any of aspects 35 to 38, wherein the expression data comprises gene expression data, proteomics expression data or a combination thereof.

40. The method of aspect 39, wherein the gene expression data comprises microarray data.

41. The method of aspect 40, wherein the microarray data comprises mRNA expression data.

42. The method of any of aspects 35 to 41, wherein the training comprises selecting target antigens that are transmembrane polypeptides.

43. The method of any of aspects 35 to 42, further comprising comparing the output of the expression data to a reference dataset.

44. The method of aspect 43, wherein the reference dataset comprises a RNAseq dataset.

45. The method of any of aspects 35 to 44, wherein applying the algorithm is performed iteratively.

46. An in vitro or ex vivo genetically modified cytotoxic immune cell, wherein the cytotoxic immune cell is genetically modified to produce a first polypeptide that recognizes a first cell surface antigen present on the surface of a target cancer cell and a second polypeptide that recognizes a second cell surface antigen present on the surface of a non-target cell.

47. The genetically modified cytotoxic immune cell of aspect 46, wherein the non-target cell is a cell of a normal tissue.

48. The genetically modified cytotoxic immune cell of aspect 47, wherein the normal tissue is brain tissue.

49. The genetically modified cytotoxic immune cell of aspects 47 or 48, wherein the second cell surface antigen is selected from the group consisting of: OPALIN, TMEM235, GABRA1, KCNJ9, GRM3, SEZ6, NTSR2, KCNK4, SLCO1A2, SLC24A2, MOG, GABRG1, GABRG2, CNTNAP4, DSCAM, CACNG3, CRB1, CDH10, HRH3, GRIK1, SLC39A12, GPR158, CACNG2, SYT3, HTR5A, CACNG7, GPR37L1, LRRTM3, GLRA2, CHRNB2, KCNQ2, JPH3, GPR19, ADCY8, SPOCK3, SLC32A1, OPCML, GABRA3, GRM5, SCN1A, SLC5A11, KCNC1, SLC12A5, GRM4, GRM1, GRIA4, MEGF11, CACNA1B, LYPD1, GRID2, SCN2A, NKAIN2, UNC5A, SLC4A10, TMEFF2, CSMD3, PPAPDC1A, HAPLN4, GPR85, ANTXR2, CACNG4, CSPG5, KCNK10, CHRNA4, CNTNAP2, KCNJ10, GABRB2, GRIN1, CRB2, SHISA7, NKAIN4, HTR2C, CACNG8, NRG3, ABCG4, CDH8, GABRD, KIRREL3, GABRB1, KCNA2, CDH20, IGDCC3, KCNJ6, CNIH2, KCNK12, CDH18, CSMD2, SYT4, OR2L13, CDH9, GABRA2, KCNF1, MAG, CALN1, GRIN2B, GRM7, VSTM2A, GPR61, OMG, KCNA1, GPR83, ATP8A2, GABBR2, GPR12, TRPM3, SLC8A3, KCND2, GSG1L, SLC30A10, ASTN1, GPR179, LRFN2, CACNA1E, CALY, SLC6A15, KIAA0319, SYT6, PTPRR, KCTD8, GPR22, SLC4A8, LAMP5, MEGF10, FXYD7, KCNK9, SLC1A6, MLC1, OPRK1, ATP2B2, ACSL6, THBD, PTPRT, PCDHGC4, CLDN10, KCNV1, LPPR3, SLCO1C1, PCDH8, ANO4, LRRTM4, PCDH15, CCKBR, GABRA5, SLC6A12, GRIN2A, SLC1A2, SLC43A1, KCNC2, ELFN2, ATP7A, GRIK4, LRRC55, HCN2, NKAIN1, DPP10, AJAP1, NPFFR1, TRPC3, TGFBI, SLC6A7, GABRA4, SLC13A5, GRIN2C, HCN1, SLC26A8, PPIC, NETO1, TNFRSF10B, CDH22, SLC6A13, DISP2, SLC6A11, CD93, EPHA10, PHLDB2, OXTR, WNT7A, GYPC, KCNA4, PCDHAC2, HGFAC, DRD1, SHISA9, SCN8A, ICAM1, PIRT, A4GALT, MRGPRF, CD248, CD58, CD44, EPHA2 and PROCR.

50. The genetically modified cytotoxic immune cell of any of aspects 46 to 49, wherein the cytotoxic immune cell is further genetically modified to produce a third polypeptides that recognizes a third cell surface antigen, wherein the third cell surface antigen is present on the surface of a second non-target cell.

51. The genetically modified cytotoxic immune cell of aspect 50, wherein the second non-target cell is a cell of a second normal tissue.

52. The genetically modified cytotoxic immune cell of aspect 51, wherein the second normal tissue is cardiac tissue.

53. The genetically modified cytotoxic immune cell of aspect 52, wherein the third cell surface antigen is selected from the group consisting of: GJA3, HCN4 and BMP10.

54. The genetically modified cytotoxic immune cell of any of aspects 46 to 53, wherein the first cell surface antigen is selected from the group consisting of: GD2 (B4GALNT1), MAGEA1, MAGEA3 and MART1 (MLANA).

55. The genetically modified cytotoxic immune cell of any of aspects 46 to 54, wherein the first and second polypeptides each comprise an antigen-triggered polypeptide.

56. The genetically modified cytotoxic immune cell of any of aspects 46 to 55, wherein the cytotoxic immune cell is a cytotoxic T cell or a natural killer cell.

57. The genetically modified cytotoxic immune cell of any of aspects 46 to 55, wherein the target cell is a melanoma.

58. A method of killing a target cancer cell in an individual, the method comprising administering to the individual an effective number of the genetically modified cytotoxic immune cell of any of aspects 46 to 57, wherein said genetically modified cytotoxic immune cell kills the target cancer cell in the individual.

59. The method of aspect 58, wherein the genetically modified cytotoxic immune cell does not substantially kill the non-target cell.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.

Example 1: Use of an AND-NOT Gate Antigen Pair as Target Antigens

CD8⁺ T cells are isolated from a patient with a liposarcoma. The CD8⁺ T cells are genetically modified with: a) a recombinant expression vector comprising a nucleotide sequence encoding a synNotch polypeptide that includes: i) a single-chain Fv (scFv) that is specific for an EVA1B polypeptide; and ii) an intracellular domain comprising a transcription activator; and b) a recombinant expression vector comprising a nucleotide sequence encoding an iCAR that comprises an antigen-binding portion (e.g., a scFv) specific for an ITGA6 polypeptide. From 10⁶ to 10⁹ genetically modified CD8⁺ T cells are administered to the patient in a single dose intravenously.

Example 2: Use of an AND Gate Antigen Pair as Target Antigens

CD8⁺ T cells are isolated from a patient with a glioblastoma. The CD8⁺ T cells are genetically modified with: a) a recombinant expression vector comprising a nucleotide sequence encoding a synNotch polypeptide that includes: i) an scFv that is specific for a PTPRZ1 polypeptide; and ii) an intracellular domain comprising a transcription activator; and b) a recombinant expression vector comprising a nucleotide sequence encoding a CAR that comprises an antigen-binding portion (e.g., a scFv) specific for a FOLR2 polypeptide. From 10⁶ to 10⁹ genetically modified CD8⁺ T cells are administered to the patient in a single dose intravenously. Upon binding of the genetically modified CD8⁺ T cells to a PTPRZ1 present on the surface of a glioblastoma in the patient, the transcription activator is released from the synNotch polypeptide. The CAR is encoded by a nucleotide sequence that is operably linked to a promoter element that is controlled by the released transcription activator. The released transcription activator induces expression of the CAR in the genetically modified CD8⁺ T cells. The CAR binds to a FOLR2 polypeptide present on the surface of the glioblastoma in the patient.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. 

1. An in vitro or ex vivo genetically modified cytotoxic immune cell, wherein the cytotoxic immune cell is genetically modified to produce two different polypeptides that recognize two different cell surface antigens, wherein at least one of the two different cell surface antigens is present on the surface of a target cancer cell.
 2. The genetically modified cytotoxic immune cell of claim 1, wherein the two different polypeptides comprise: a) a first antigen-triggered polypeptide that binds specifically to a first target cell surface antigen present on a target cancer cell; and b) a second antigen-triggered polypeptide that binds specifically to a second target cell surface antigen.
 3. The genetically modified cytotoxic immune cell of claim 1, wherein the cytotoxic immune cell is a cytotoxic T cell or a natural killer cell.
 4. The genetically modified cytotoxic immune cell of claim 1, wherein the immune cell is activated to kill a target cancer cell only when the target cancer cell expresses both of the two different cell surface antigens on its cell surface.
 5. The genetically modified cytotoxic immune cell of claim 2, wherein the cytotoxic immune cell: a) is activated to kill a target cancer cell that expresses the first target cell surface antigen, but not the second target cell surface antigen, on its cell surface; and b) is inhibited from killing a non-cancerous cell if the non-cancerous cell expresses both the first target cell surface antigen and the second target cell surface antigen on its cell surface.
 6. The genetically modified cytotoxic immune cell of claim 2, wherein the first antigen-triggered polypeptide is a synNotch receptor and the second antigen-triggered polypeptide is a chimeric antigen receptor (CAR); wherein the first antigen-triggered polypeptide is a synNotch receptor and the second antigen-triggered polypeptide is a T cell receptor (TCR); wherein the first antigen-triggered polypeptide is a CAR and the second antigen-triggered polypeptide is an inhibitory CAR (iCAR); wherein the first antigen-triggered polypeptide is a CAR and the second antigen-triggered polypeptide is a synNotch receptor; wherein the first antigen-triggered polypeptide is a synNotch receptor and the second antigen-triggered polypeptide is a synNotch receptor: or wherein the first antigen-triggered polypeptide is a synNotch receptor and activation of the synNotch receptor induces expression of an immune inhibitory polypeptide. 7-16. (canceled)
 17. A method of killing a target cancer cell in an individual, the method comprising administering to the individual an effective number of the genetically modified cytotoxic immune cell of claim 1, wherein said genetically modified cytotoxic immune cell kills the target cancer cell in the individual.
 18. The method of claim 17, wherein the target cancer cell is an AML cell, an anaplastic lymphoma cell, an astrocytoma cell, a B-cell cancer cell, a breast cancer cell, a colon cancer cell, an ependymoma cell, an esophageal cancer cell, a glioblastoma cell, a glioma cell, a leiomyosarcoma cell, a liposarcoma cell, a liver cancer cell, a lung cancer cell, a mantle cell lymphoma cell, a melanoma cell, a neuroblastoma cell, a non-small cell lung cancer cell, an oligodendroglioma cell, an ovarian cancer cell, a pancreatic cancer cell, a peripheral T-Cell lymphoma cell, a renal cancer cell, a sarcoma cell or a stomach cancer cell.
 19. A system for killing a target cancer cell, the system comprising: a) a first antigen-triggered polypeptide that binds specifically to a first target antigen present on the target cancer cell, or a first nucleic acid comprising a nucleotide sequence encoding the first antigen-triggered polypeptide; and b) a second antigen-triggered polypeptide that binds specifically to a second target antigen, or a second nucleic acid comprising a nucleotide sequence encoding the second antigen-triggered polypeptide.
 20. The system of claim 19, wherein the first antigen-triggered polypeptide is a synNotch polypeptide, and the second antigen-triggered polypeptide is a chimeric antigen receptor (CAR); wherein the first antigen-triggered polypeptide is a synNotch polypeptide and the second antigen-triggered polypeptide is an inhibitory CAR (iCAR); wherein the first antigen-triggered polypeptide is a synNotch polypeptide and the second antigen-triggered polypeptide is one polypeptide chain of a split CAR; wherein the first antigen-triggered polypeptide is a synNotch polypeptide and the second antigen-triggered polypeptide is a T-cell receptor; wherein the first antigen-triggered polypeptide is a synNotch polypeptide and the second antigen-triggered polypeptide is an immunoinhibitory polypeptide; wherein the first antigen-triggered polypeptide is a first synNotch polypeptide and the second antigen-triggered polypeptide is a second synNotch polypeptide: or wherein the first antigen-triggered polypeptide induces production of an antibody or cytokine upon binding to the first target antigen. 21-22. (canceled)
 23. The system of claim 19, wherein the first target antigen and the second target antigen are both present on the surface of a target cancer cell; or wherein a) the first target antigen and the second target antigen are both present on the surface of a non-cancerous cell, and b) the first target antigen, but not the second target antigen, is present on the surface of a target cancer cell.
 24. (canceled)
 25. The system of claim 19, wherein the first target antigen and the second target antigen are selected from a target antigen pair depicted in FIG. 1 or FIG. 9-14 . 26-30. (canceled)
 31. A method of killing a target cancer cell in an individual, the method comprising: a) introducing the system of claim 19 into a cytotoxic T cell in vitro or ex vivo, generating a modified cytotoxic T cell; and b) administering the modified cytotoxic T cell to the individual.
 32. (canceled)
 33. The method of claim 31, wherein the modified cytotoxic T cell is activated to kill the target cancer cell only when the target cancer cell expresses both the first target antigen and the second target antigen on its cell surface; or wherein the modified cytotoxic T cell a) is activated to kill a target cancer cell that expresses the first target cell surface antigen, but not the second target cell surface antigen, on its cell surface; and b) is inhibited from killing a non-cancerous cell if the non-cancerous cell expresses both the first target cell surface antigen and the second target cell surface antigen on its cell surface.
 34. (canceled)
 35. A method of identifying a combination of target antigens for targeting a cancer cell, the method comprising: a) generating a training set of target antigen parameters by determining, from a training set of expression data, pairs of antigens that discriminate between cancer cells and non-cancerous cells, thereby generating an algorithm; b) applying the algorithm to a test set of expression data, to generate a combination of target antigens.
 36. The method of claim 35, wherein step (a) comprises selecting pairs of target antigens that comprise: i) a first target antigen that is expressed at a high level on cancer cells of a selected cancer cell type; and ii) a second target antigen that is expressed at a high level on cancer cells of the selected cancer cell type; or i) a first target antigen that is expressed at a high level on cancer cells of a selected cancer cell type and on non-cancerous cells of the same cell type; and ii) a second target antigen that is expressed at a high level on non-cancerous cells of the same cell type but not on cancer cells of the same cell type.
 37. The method of claim 35, wherein the algorithm comprises one or more of an F1 cutoff, a precision cutoff, a recall cutoff, or combination thereof.
 38. The method of claim 35, wherein step (a) comprises training a computer software with the training set, and wherein step (b) comprises applying the algorithm using the trained computer software. 39-45. (canceled)
 46. The in vitro or ex vivo genetically modified cytotoxic immune cell of claim 1, wherein the two different polypeptides comprise a first polypeptide that recognizes a first cell surface antigen present on the surface of a target cancer cell and a second polypeptide that recognizes a second cell surface antigen present on the surface of a non-target cell.
 47. The genetically modified cytotoxic immune cell of claim 46, wherein the non-target cell is a cell of a normal tissue.
 48. The genetically modified cytotoxic immune cell of claim 47, wherein the normal tissue is brain tissue.
 49. The genetically modified cytotoxic immune cell of claim 47, wherein the second cell surface antigen is selected from the group consisting of: OPALIN, TMEM235, GABRA1, KCNJ9, GRM3, SEZ6, NTSR2, KCNK4, SLCO1A2, SLC24A2, MOG, GABRG1, GABRG2, CNTNAP4, DSCAM, CACNG3, CRB1, CDH10, HRH3, GRIK1, SLC39A12, GPR158, CACNG2, SYT3, HTR5A, CACNG7, GPR37L1, LRRTM3, GLRA2, CHRNB2, KCNQ2, JPH3, GPR19, ADCY8, SPOCK3, SLC32A1, OPCML, GABRA3, GRM5, SCN1A, SLC5A11, KCNC1, SLC12A5, GRM4, GRM1, GRIA4, MEGF11, CACNA1B, LYPD1, GRID2, SCN2A, NKAIN2, UNC5A, SLC4A10, TMEFF2, CSMD3, PPAPDC1A, HAPLN4, GPR85, ANTXR2, CACNG4, CSPG5, KCNK10, CHRNA4, CNTNAP2, KCNJ10, GABRB2, GRIN1, CRB2, SHISA7, NKAIN4, HTR2C, CACNG8, NRG3, ABCG4, CDH8, GABRD, KIRREL3, GABRB1, KCNA2, CDH20, IGDCC3, KCNJ6, CNIH2, KCNK12, CDH18, CSMD2, SYT4, OR2L13, CDH9, GABRA2, KCNF1, MAG, CALN1, GRIN2B, GRM7, VSTM2A, GPR61, OMG, KCNA1, GPR83, ATP8A2, GABBR2, GPR12, TRPM3, SLC8A3, KCND2, GSG1L, SLC30A10, ASTN1, GPR179, LRFN2, CACNAlE, CALY, SLC6A15, KIAA0319, SYT6, PTPRR, KCTD8, GPR22, SLC4A8, LAMP5, MEGF10, FXYD7, KCNK9, SLC1A6, MLC1, OPRK1, ATP2B2, ACSL6, THBD, PTPRT, PCDHGC4, CLDN10, KCNV1, LPPR3, SLCO1C1, PCDH8, ANO4, LRRTM4, PCDH15, CCKBR, GABRA5, SLC6A12, GRIN2A, SLC1A2, SLC43A1, KCNC2, ELFN2, ATP7A, GRIK4, LRRC55, HCN2, NKAIN1, DPP10, AJAP1, NPFFR1, TRPC3, TGFBI, SLC6A7, GABRA4, SLC13A5, GRIN2C, HCN1, SLC26A8, PPIC, NETO1, TNFRSF10B, CDH22, SLC6A13, DISP2, SLC6A11, CD93, EPHA10, PHLDB2, OXTR, WNT7A, GYPC, KCNA4, PCDHAC2, HGFAC, DRD1, SHISA9, SCN8A, ICAM1, PIRT, A4GALT, MRGPRF, CD248, CD58, CD44, EPHA2 and PROCR. 50-59. (canceled) 